Intracranial botulinum toxin therapy for focal epilepsy

ABSTRACT

Methods for treating and/or curing epilepsy by intracranial administration of a botulinum toxin.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.10/421,504, filed Apr. 22, 2003, which is a continuation in part of U.S.application Ser. No. 09/903,849, filed Jul. 12, 2001, now abandoned,which is a divisional of 09/596,306, filed Jun. 14, 2000, now U.S. Pat.No. 6,306,403.

BACKGROUND

The present invention relates to methods for treating movementdisorders. In particular, the present invention relates to methods fortreating epilepsy by intracranial administration of a botulinum toxin.

Intracranial Drug Delivery

A major impediment to therapeutic treatment of a neurodegenerativedisease, such as various movement disorders, is the blood-brain barrierwhich significantly limits penetration of the brain by even smallmolecules from the bloodstream upon peripheral administration of apharmaceutical. To circumvent the blood-brain barrier direct infusion ofvarious bioactive substances has been carried out. Most clinicalexperience is with intraventricular (i.e. into a cerebral-spinal fluid[CSF] filled ventricle of the brain) drug delivery. Thus, ventricularinfections have been treated by direct infusion of antibiotic.Additionally, intraventricular infusion: of baclofen to treatspasticity; various chemotherapeutics, radiolabelled antibodies, andcytokines to treat brain tumors; cholinergic agonists and Nerve GrowthFactor (NGF) to treat Alzheimer's disease, and; dopamine to treatParkinson's disease is known. Unfortunately, there is a brain-CSFbarrier such that penetration of drugs into brain tissue from CSF issuboptimal. Intraventricular drug delivery has therefore been met withlimited success in the treatment of, for example, solid tumors,neurodegenerative diseases (such as movement disorders) and otherintraparenchymal pathology.

The drawbacks and deficiencies of intraventricular drug delivery has ledto increasing interest in direct infusion of drugs into brainparenchyma. Administration of therapeutic bioactive substances tovarious brain sites of interest has be achieved with reproduciblesubmillimeter precision using modern stereotactic techniques. Thus,intraparenchymal infusion of lidocaine, muscimol and NGF have been usedto treat Parkinson's disease, and intraparenchymal infusion of KCL hasbeen used to treat epilepsy. The most widespread use of directintracerebral clinical administration of a bioactive substance has beento treat brain tumors, which has included gene therapy by delivery ofone or more therapeutic genes into brain tumor cells. See e.g. KaplittM. G. et al., Surgical drug delivery for neurodegenerative diseases.Clin Neurosurg 48; 127-144: 2001. The localized microinjection oflidocaine and muscimol has been found to selectively inactivate focalneuronal activity in the subthalamic nucleus of Parkinson's diseasepatients (Levy R. et al., Lidocaine and muscimol microinjections insubthalamic nucleus reverse Parkinsonian symptoms, Brain 2001 October;124(Pt 10):2105-18). The effect is transient and is directlyattributable to intrinsic activity and pharmacological half-lives ofthese agents.

Additionally, patients with malignant gliomas have been treated with achimeric toxin composed of the cytokine, IL-4, and Pseudomonas exotoxin,administered through stereotactic catheter implantation into tumorsthrough small-twist drill holes. Despite varying volumes of infusion, noprofound neural or systemic toxicity resulted. Rand R. W. et al.,Intratumoral administration of recombinant circularly permutedinterleukin-4-Pseudomonas exotoxin in patients with high-grade glioma,Clin Cancer Res 6; 2157-2165: 2000.

Thus, precise intracranial therapeutic delivery of bioactivemacromolecules can be achieved through stereotactic methodologies. Whena molecule is introduced into the extracellular space in brain tissue,because of the narrowness and irregularity of channels, it diffusesthrough the tissue at a rate which is at least twelve times slower thandiffusion of the molecule through agar. Penn R., The future of CNSinfusion systems, chapter 218, pages 2073-2076 of Gildenberg, P.,Textbook of Stereotactic and Functional Neurosurgery, McGraw-Hill(1998). Due to take up by cells, binding of receptors or extracellularmatrix, enzymatic degradation, or elimination by the vascular system itis unusual for a bioactive molecule applied as a point source to diffusein brain tissue more than a few millimeters from its' site ofadministration. Ibid. For functional stereotactic surgeons the type ofapplications most suited for intraparenchymal drug application are thosein which a small lesion or local electrical stimulation has alreadyproved effective, such as for the treatment of tremor. Thus, it has beenpostulated that the use of selective neurotransmitter agents orantagonists may be more effective to inhibit specific neurons than iscurrent therapies of deliberately making or inducing a lesion (i.e. byradiation, thermal, cryo or electrical ablation or surgical incision)that indiscriminately destroys neurons and axons. Ibid.

Movement Disorders

A movement disorder is a neurological disturbance that involves one ormore muscles or muscle groups. Movement disorders include Parkinson'sdisease, Huntington's Chorea, progressive supranuclear palsy, Wilson'sdisease, Tourette's syndrome, epilepsy, and various chronic tremors,including essential tremor, tics and dystonias. Different clinicallyobserved movement disorders can be traced to the same or similar areasof the brain. For example, abnormalities of basal ganglia (a largecluster of cells deep in the hemispheres of the brain) are postulated asa causative factor in diverse movement disorders.

Tremors are characterized by abnormal, involuntary movements. Anessential tremor is maximal when the body part afflicted (often an armor hand) is being used, for example when attempts at writing or finecoordinated hand movements are made. Typical chemotherapy is use of thedrug propranolol (Inderal) which has the side effects of low bloodpressure and heart rate changes. A resting tremor is common inParkinson's disease and in syndromes with Parkinsonian features. Aresting tremor is maximal when the extremities are at rest. Often, whena patient attempts fine movement, such as reaching for a cup, the tremorsubsides. Systemic anticholinergic medications have been used with somesuccess.

Dystonias are involuntary movement disorders characterized by continuedmuscular contractions which can result in twisted contorted posturesinvolving the body or limbs. Causes of dystonia include biochemicalabnormalities, degenerative disorders, psychiatric dysfunction, toxins,drugs and central trauma. Thalamotomy and/or subthalamotomy or campotomyare currently the preferred neurosurgical procedures to treat dystonia,and are carried out with techniques and brain targets similar to thesurgical treatment of Parkinson's disease. Tasker R., Surgical Treatmentof the Dystonias, chapter 105, pages 1015-1032, in Gildenberg P. L. etal., Textbook of Stereotactic and Functional Neurosurgery, McGraw-Hill(1998).

Particular dystonias can include spasmodic torticolis, blepharospasm andwriter's cramp. Spasmodic torticollis is a syndrome that usually affectsadults, and involves the involuntary turning of the neck to one side.Some individuals may not even notice initially that the head and neckare turned. Blepharospasm is an involuntary movement which involvesintermittent forceful closure of the eyelids. Writer's cramp is acramping abnormal posture which develops when one is writing, orperforming other actions with the hands. Symptoms may progress toinvolve the arm and shoulder.

Tic disorders (including Tourette's) are usually very rapid, short livedstereotyped repeated movements. The more common tics involve the motorsystems, or are vocal in nature. Motor tics often involve the eyelids,eyebrows or other facial muscles, as well as the upper limbs. Vocal ticsmay involve grunting, throat clearing, coughing or cursing. Individualswith tic disorders will often describe a strong urge to perform theparticular tic, and may actually feel a strong sense of pressurebuilding up inside of them if the action is not performed. For example,a motor tic that may involve the abrupt movement of one of the arms maybe controllable for a short period of time if the affected person sitson his hands; however, the almost irresistible urge to do the actionoften takes over and result in the tic action.

Tourette's syndrome is a tic disorder which often begins in childhood oradolescence and is much more common in males. There are both multiplemotor tics, as well as vocal tics present. The tics often change frominvolvement of one body part to another, and the disease often getsbetter and worse intermittently, with periods of almost minimalactivity, and other times when some patients have difficultyfunctioning. Other neurobehavioral difficulties often accompany thesyndrome. These include attention deficit hyperactivity disorder (ADHD)and obsessive-compulsive disorder. Treatment of most tic disordersemploys the use of medications that decrease the amount of dopamine inthe brain, such as dopamine antagonists. Unfortunately these drugs areassociated with side effects such as other movement disorders, includingParkinsonism (stiffness, slow movement and tremors). In addition toTourette's syndrome, tics may be associated with head injury, carbonmonoxide poisoning, stroke, drug use and mental retardation.

Progressive supranuclear palsy is a movement disorder in which patientshave significant difficulty moving their eyes vertically (up and down)initially, followed by all eye movements become limited(opthalmoplegia). Patients can also develop dementia, rigidity,bradykinesia (slow movements) and a propensity for falls.

Huntington's chorea is a genetically inherited disorder that has bothneurological and psychiatric features. Most cases develop when peopleare in their forties or fifties, but early and late onset is alsopossible. The disease may begin with either the neurological or mentalstatus changes. The neurological symptoms may vary, but include chorea.Chorea (derived from a Greek word meaning, “to dance”) is a series ofmovements that is dance-like, jerky, brief, and moves from one part ofthe body to another. Clumsiness, fidgetiness and jumpiness may alsooccur. Facial movements, especially around the jaw, may occur. There isoften difficulty with walking and posture. The psychiatric symptoms maypresent as paranoia, confusion, or personality changes. As the diseaseprogresses, a significant dementia develops. MRI brain imaging may showatrophy (shrinkage) of a portion of the basal ganglia (involved inmovement) that is known as the caudate nucleus.

Wilson's disease is a disorder that involves the nervous system andliver function. The neurological problems include tremors,incoordination, falling, slurred speech, stiffness and seizures.Psychiatric problems can occur and patients can develop severe liverdamage if this affliction is untreated. Elevated copper andceruloplasmin levels are diagnostic.

Unfortunately, a movement disorder, including those set forth above, canbecome resistant to drug therapy. Drug resistant tremors can includeresting tremors, such as can occur in Parkinson's disease, and actiontremors, such as essential tremor, multiple sclerosis tremors, posttraumatic tremors, post hemiplegic tremors (post stroke spasticity),tremors associated with neuropathy, writing tremors and epilepsy.

Parkinson's Disease

Parkinson's disease is a movement disorder of increasing occurrence inaging populations. Parkinson's disease is a common disabling disease ofold age affecting about one percent of the population over the age of 60in the United States. The incidence of Parkinson's disease increaseswith age and the cumulative lifetime risk of an individual developingthe disease is about 1 in 40. Symptoms include pronounced tremor of theextremities, bradykinesia, rigidity and postural change. A perceivedpathophysiological cause of Parkinson's disease is progressivedestruction of dopamine producing cells in the basal ganglia whichcomprise the pars compartum of the substantia nigra, a basal nucleilocated in the brain stem. Loss of dopamineric neurons results in arelative excess of acetylcholine. Jellinger, K. A., Post Mortem Studiesin Parkinson's Disease—Is It Possible to Detect Brain Areas For SpecificSymptoms?, J Neural Transm 56 (Supp); 1-29:1999.

Parkinson's disease is a progressive disorder which can begin with mildlimb stiffness and infrequent tremors and progress over a period of tenor more years to frequent tremors and memory impairment, touncontrollable tremors and dementia.

Drugs used to treat Parkinson's disease include L-dopa, selegiline,apomorphine and anticholinergics. L-dopa (levo-dihydroxy-phenylalanine)(sinemet) is a dopamine precursor which can cross the blood-brainbarrier and be converted to dopamine in the brain. Unfortunately, L-dopahas a short half life in the body and it is typical after long use (i.e.after about 4-5 years) for the effect of L-dopa to become sporadic andunpredictable, resulting in fluctuations in motor function, dyskinesiasand psychiatric side effects. Additionally, L-dopa can cause B vitamindeficiencies to arise.

Selegiline (Deprenyl, Eldepryl) has been used as an alternative toL-dopa, and acts by reducing the breakdown of dopamine in the brain.Unfortunately, Selegiline becomes ineffective after about nine months ofuse. Apomorphine, a dopamine receptor agonist, has been used to treatParkinson's disease, although is causes severe vomiting when used on itsown, as well as skin reactions, infection, drowsiness and somepsychiatric side effects.

Systemically administered anticholinergic drugs (such as benzhexyl andorphenedrine) have also been used to treat Parkinson's disease and actby reducing the amount of acetylcholine produced in the brain andthereby redress the dopamine/acetylcholine imbalance present inParkinson's disease. Unfortunately, about 70% of patients takingsystemically administered anticholinergics develop seriousneuropsychiatric side effects, including hallucinations, as well asdyskinetic movements, and other effects resulting from wideanticholinergic distribution, including vision effects, difficultyswallowing, dry mouth and urine retention. See e.g. Playfer, J. R.,Parkinson's Disease, Postgrad Med J, 73; 257-264:1997 and Nadeau, S. E.,Parkinson's Disease, J Am Ger Soc, 45; 233-240:1997.

Before the introduction of L-dopa in 1969, stereotactic surgery offeredone of the few effective treatments for Parkinson's disease. Thesignificant known deficiencies and drawbacks associated with therapeuticdrugs to treat Parkinson's disease, including the long term limitationsof L-dopa therapy have led to renewed interest in neurosurgicalintervention. Unilateral stereotactic thalamotomy has proven to beeffective for controlling contralateral tremor and rigidity, but carriesa risk of hemiparesis. Bilateral thalamotomy carries an increased riskof speech and swallowing disorders resulting. Stereotactic pallidotomy,surgical ablation of part of the globus pallidus (a basal ganglia), hasalso be used with some success. Aside from surgical resection, highfrequency stimulating electrodes placed in the ventral intermedialisnucleus has been found to suppress abnormal movements in some cases. Avariety of techniques exist to permit precise location of a probe,including computed tomography and magnetic resonance imaging.Unfortunately, the akinesia, speech and gait disorder symptoms ofParkinson's disease are little helped by these surgical procedures, allof which result in destructive brain lesions.

Epilepsy

Epilepsy is the most common serious neurological disorder (Shorvon, S.,Epidemiology, classification, natural history, and genetics of epilepsy,Lancet 1990 Jul. 14; 336(8707):93-6; McNamara J., The neurobiologicalbasis of epilepsy, Trends Neurosci 1992 October; 15(10):357-9. A seizureis a neurological dysfunction which results from abnormal, excessive,hypersynchronous discharges from an aggregate of central nervous systemneurons. A seizure can be manifested behaviorally (if motor systems areinvolved) or electrographically. Epilepsy describes a condition in whicha person has recurrent seizures due to a chronic, underlying process.Although there are various epilepsy syndromes in which the clinical andpathologic characteristics differ the common underlying etiology isneuronal hyperexcitability. Thus, epilepsy encompasses disorders ofcentral nervous system (CNS) hyperexcitability, characterized bychronic, recurrent, paroxysmal changes in neurological function that canbe categorized according to electroencephalographic and clinicalpresentation (Dichter M., Basic mechanisms of epilepsy: targets fortherapeutic intervention, Epilepsia 1997; 38 Suppl 9:S2-6).

Excluding neonatal febrile seizures, the estimated occurrence ofepilepsy in the general population is about 0.5%-1% (Barnes D., Debateabout epilepsy: what initiates seizures?, Science 1986 Nov. 21;234(4779):938-40, erratum in Science 1987 Jan. 2; 235(4784):16; RogawskiM., et al., Antiepileptic drugs: pharmacological mechanisms and clinicalefficacy with consideration of promising developmental stage compounds,Pharmacol Rev 1990 September; 42(3):223-86). Severe, penetrating headtrauma is associated with up to a 50% risk of leading to epilepsy. Othercauses of epilepsy include stroke, infection and genetic susceptibility.

While recurrent seizures are a hallmark of epilepsy, isolated,nonrecurrent seizures can occur in otherwise healthy individuals for avariety of reasons, such as poisoning, and such individuals are notconsidered to have epilepsy (Dichter 1997, Ibid). Epileptic seizures arebroadly categorized into two groups: focal (partial) and generalizedseizures. Focal seizures arise from abnormal activity of a limited groupof neurons in cortical or subcortical regions of the brain. Theunderlying structural abnormality or lesion can develop as a result ofbirth injury, head trauma, tumor, abscess, infarction, vascularmalformation or genetic disease (Dichter 1997, Ibid). The location ofthe focal activity can be identified by the clinical seizurepresentation or may be cryptic. Equivalently, the active focus may notinvolve the lesion itself but may arise in adjacent or distant (butconnected) neuronal populations, supporting the hypothesis of plasticsynaptic reorganization underlying focal hyperexcitability. See e.g.Prince D. A., Epileptogenic neurons and circuits. In: Jasper's BasicMechanisms of the Epilepsies, Third Edition (1999), Delgado-Escueta A.V., et al., editors), Advances in Neurology 79: 665-684.

Focal seizures are termed “simple” if there is no apparent change inconsciousness, otherwise they are termed “complex”. Complex focalseizures involve the temporal lobe and limbic system, and are the mostcommon manifestation of epilepsy in adults. Focal seizures that spreadto become bilateral electrographically, with concomitant loss ofconsciousness and with or without motor manifestations, are said to besecondarily generalized. Primary generalized seizures initiate withbilateral electrographic activity, loss of consciousness, and with orwithout motor convulsions. Focal epilepsy can involve almost any part ofthe brain and usually results from a localized lesion of functionalabnormality. One type of focal epilepsy is the psychomotor seizure.Current therapy for focal epilepsy includes use of an EEG to localizeabnormal spiking waves originating in areas of organic brain diseasethat predispose to focal epileptic attacks, followed by surgicalexcision of the focus to prevent future attacks.

While the etiology of epilepsy can be multiple, the pathophysiology ofepilepsy (in terms of the generation of synchronized neuronal activity)is thought to consistently reflect both fundamental changes in basicneuronal physiological properties together with enhanced synapticplasticity. The process of epileptogenesis therefore involves a limitedrange of intrinsic cellular changes that lead to neuronal imbalances innet excitation or inhibition, or in the enhanced excitatory coupling ofneuronal aggregates (Prince 1999 ibid, and; Prince D. A., Cellularmechanisms of interictal-ictal transitions. In: Mechanisms ofEpileptogenesis. The Transition to Seizure, Dichter M. A., editor,Plenum Press, New York, 57-71 (1998)). The hallmark of epileptogenesisis the appearance of interictal (between seizure) bursts or discharges.This pattern of activity is observed on the EEG record as brief (80-200millisecond), large, sharp spikes against an otherwise normal backgroundof activity (McCormick and Contreras 2001, supra). These periodic EEGspikes correlate to prolonged cellular depolarizations (paroxysmaldepolarization shift, PDS), while the quiescent periods reflect phasesof cellular hyperpolarization.

Thus it is believed that distinct neural circuitry underlying theinitiation and propagation of seizures can be identified for each typeof epileptic seizures because seizures are manifestations of abnormalactivity in neuronal networks that normally are engaged in routinephysiological processes (Gale K., Focal trigger zones and pathways ofpropagation in seizure generation. In: Epilepsy: Models, Mechanism andConcepts, Schwartzkroin P. A., editor, Cambridge University Press, U.K.,pages 48-93 (1993)).

While there may be no dedicated “seizure circuit” within the brain,seizure propagation clearly results from, and depends upon, the specificneuroanatomy of the interconnected neuronal circuitry. It is wellestablished that some parts of the brain, such as the limbic system, aremore susceptible to epileptogenesis and seizure propagation than areother areas, such as the neocortex. The hippocampus (a part of thelimbic system) has been extensively studied, as much for its orderly andaccessible cellular architecture as for its tendency to be becomeepileptogenic when provided an appropriate stimulus, both in vitro andin vivo. The epileptogenic hippocampus displays significant synapticreorganization and changes in plasticity that potentiatehyperexcitability.

Mossy fiber sprouting, secondary to loss of target cells in Ammon's hornarea CA3 of the hippocampus, and subsequent establishment of synapses inthe inner molecular layer of the normally hypoexcitable dentate gyrus(fascia dentata), results in recurrent (feedback) excitation (Sutula T.P., Sprouting as an underlying cause of hyperexcitability inexperimental models and in the human epileptic temporal lobe. In:Epilepsy: Models, Mechanism and Concepts, Schwartzkroin P. A. editor,Cambridge University Press, U.K., pages 304-322 (1993)). Thissynchronized hippocampal activity directly results from phasicimbalances between excitatory and inhibitory neuronal populations. Thehippocampus, together with the amygdala, have been implicated in humantemporal lobe epilepsy, an often intractable condition and the mostcommon epileptic disorder in adults. Cellular degeneration in thehippocampus (termed hippocampal sclerosis), and compensatory changes inwiring, are a prime cause of hyperexcitability and a rationale forresective surgery in the treatment of temporal lobe epilepsy. Aspreviously noted, the neocortex is less vulnerable to seizurepropagation from local foci as compared to the limbic system. Thisself-limiting capacity may arise from the inherent characteristic ofselective presynaptic depression of excitatory transmission underconditions of high frequency firing (Galaretta M. et al.,Frequency-dependent synaptic depression and the balance of excitationand inhibition in the neocortex, Nature Neurosci 1(7); 587-594: 1998),as would occur during seizure propagation. Changes in the connectivityand communication between neuronal cells (neural plasticity) have beenimplicated in the pathophysiology of epilepsy. Synaptic plasticityrefers to characteristic activity-dependent changes in synaptic efficacythat may either produce enhancement (long term potentiation, LTP) orinhibition (long term depression). The observed changes may reflectaltered functionality at presynaptic and postsynaptic locations.Presynaptic changes, which involve alterations in the kinetics ofsynaptic vesicle recycling, affect the rate of neurotransmission and,therefore, of synaptic activity (Staley K., et al., Presynapticmodulation of CA3 network activity, Nat Neurosci 1998 July; 1(3):201-9,erratum in Nat Neurosci 1998 August; 1(4):331; Wang L., et al.,High-frequency firing helps replenish the readily releasable pool ofsynaptic vesicles, Nature 1998 Jul. 23; 394(6691):384-8; Zakharenko S.,et al., Visualization of changes in presynaptic function duringlong-term synaptic plasticity, Nat Neurosci 2001 July; 4(7):711-7).Synchronization of presynaptic and postsynaptic activity can alsoenhance the efficacy of synaptic transmission and facilitate LTP(Ganguly K., et al., Enhancement of presynaptic neuronal excitability bycorrelated presynaptic and postsynaptic spiking, Nat Neurosci 2000October; 3(10):1018-26). Postsynaptic changes in synaptic plasticityhave been suggested as the basis for such processes as learning andmemory and may contribute, in part, to the process of epileptogenesis.

This neuroanatomical basis for brain neuronal network connectivity, andthe mechanisms of epileptogenesis have been investigated, for example,in the kindling model of focal, temporal lobe (limbic) epilepsy. In thekindling paradigm, test animals are subjected to repeated, focalelectrical stimulation through bipolar, stereotactically-implanted depthelectrodes (Goddard G. V. et al, A permanent change in brain functionresulting from daily electrical stimulation. Exp Neurol 25(3);295-330:1969). The electrodes are typically placed in either theamygdala or dorsal hippocampus of rats. The initially subconvulsivestimulations gradually precipitate a described progression of behavioralmanifestations, which are then scored (Racine R. J., Modification ofseizure activity by electrical stimulation. II. Motor seizure,Electroencephalogr Clin Neurophysiol. 32(3); 281+−94:1972b), and whichcorrelate to the gradual electrographic appearance of afterdischarges(episodic, ictal, electrical activity in the absence of exogenousstimulus). Together with their appearance, the threshold electricalstimulus required to produce an afterdischarge gradually lowers,paralleling the seizure threshold drop seen in human epileptogenesis.The kindling process fails to develop in the absence of afterdischarges(Racine R. J., Modification of seizure activity by electricalstimulation. I. After-discharge threshold, Electroencephalogr ClinNeurophysiol. 32(3); 269-79:1972a). Eventually, the continued repeatedstimulations precipitate full motor seizures (Goddard et al, 1969,supra). Once established, the lowered seizure threshold andhyperexcitability are permanent, such that an otherwise subconvulsive(threshold) stimulus at a later time point triggers a generalized,convulsive seizure. It should be noted that the generalized seizure issimply an end point, reflecting recruitment of motor output pathways,and does not in and of itself establish the kindled phenotype. Rather,kindling reflects the whole process of population recruitment and spreadby repeated focal afterdischarge.

Antiepileptic drug (“AED”) therapy is the mainstay of treatment for mostpatients with epilepsy and a variety of drugs have been used. See e.g.,Fauci, A. S. et al., Harrison's Principles of Internal Medicine,McGraw-Hill, 14^(th) Edition (1998), page 2321. About twenty percent ofpatients with epilepsy are resistant to drug therapy despite efforts tofind an effective combination of antiepileptic drugs. Surgery can thenbe an option. Thus, patients with refractory epilepsy and intractableseizures can be candidates for surgical resection followed by up AEDtherapy. Surgery is superior to long-term drug treatment alone in themanagement of temporal lobe epilepsy (Wiebe et al, 2001, supra).Typically, video-electroencephalogram (EEG) monitoring is used tobroadly define the anatomic location of the seizure focus and tocorrelate the abnormal electrophysiological activity with behavioralmanifestations of the seizure. Scalp or scalp-sphenoidal recordings areusually sufficient for localization. Functional imaging studies such asSPECT and PET, as well as direct electrophysiological analysis (subduralor depth electrode mapping) are adjunctive tests that can help verifythe localization of an apparent epileptogenic region with an anatomicabnormality. A high resolution MRI scan is routinely used to identifystructural lesions. Once the presumed location of the seizure onset isidentified, additional studies, including neuropsychological testing andthe intracarotid amobarbital test (Wada's test) can be used to assesslanguage and memory localization and to determine the possiblefunctional consequences of surgical removal of the epileptogenic region.In some cases, the exact extent of the resection to be undertaken can bedetermined by performing cortical mapping at the time of the surgicalprocedure. This involves electrophysiologic recordings and corticalstimulation of the awake patient to identify the extent of epileptiformdisturbances and the function of the cortical regions in questions.Clearly, consideration for surgery is contingent upon the identified andimplicated substructure being resectable. The most common surgicalprocedures for patients with temporal lobe epilepsy are resection of theanteromedial temporal lobe (temporal lobectomy, which includes thenterolateralneocortex and the deeply located olderstructures-corticoamygdalohippocampectomy), or a more limited removal ofthe underlying hippocampus and amygdala (selectiveamygdalohippocampectomy. A third procedure, the selective neocorticalresection, is rarely used, although focal seizures arising fromextratemporal regions may be suppressed by a focal neocorticalresection.

While invasive, surgery has proven to be superior to long-term drugtreatment alone in the management of temporal lobe epilepsy (Wiebe S. etal, A randomized, controlled trial of surgery for temporal-lobeepilepsy, N Engl J Med 345(5): 311-8:2001). Unfortunately, about 5% ofpatients can still develop clinically significant complications fromsurgery and up to 10% of temporal lobectomy patients can still haveseizures. Kwan P. et al, Refractory epilepsy: a progressive, intractablebut preventable condition? Seizure 11: 77-84:2002). In cases wheresubcortical structures encompass, or are in proximity to, criticalfunctional areas, and thus cannot be approached with standard resectivetechniques, the treatment regimen can be a maintenance of multiple AEDsfor suppression of seizure activity (Kwan et al 2002, supra).

Stereotactic surgical procedures have been refined and aided byimprovements in current imaging techniques, permitting preciseidentification and targeting of intracranial substructures, compared tomore traditional ventriculography. MRI methods allow for clear andaccurate definition, three-dimensional spatial orientation and structurelocalization. See e.g. Landi A. et al., Accuracy of stereotacticlocalization with magnetic resonance compared to CT scan: experimentalfindings, Acta Neurochir (Wien) 143; 593-601: 2001. As compared tohistorically traditional craniotomy and resective surgery, stereotacticsurgery (as with, for example, refractive temporal lobe epilepsy) may bebetter tolerated by patients and more economical to perform. Parrent A.G. et al., Stereotactic surgery for temporal lobe epilepsy, Can J NeurolSci 27(Suppl 1); S79-S84: 2000. In addition to guiding surgicalresections, stereotactic techniques can be employed, alternatively, tospecifically target a selected structure and deliver an effectivetherapy. While all surgical procedures are invasive and irreversible,stereotactically targeted therapies may be either invasive ornoninvasive, and either irreversible or reversible.

Stereotactic radiofrequency ablation (invasive, irreversible) has beendescribed for the treatment of intractable gelastic seizures arisingfrom a hypothalamic hamartoma following MR imaging, focus localizationand reference-based guidance of the lesioning electrode to thehypothalamus. See e.g. Parrent A. G., Stereotactic radiofrequencyablation for the treatment of gelastic seizures associated withhypothalamic hamartoma: case report, J Neurosurg 91: 881-884: 1999.Similarly, and in a manner analogous to that applied to the treatment ofParkinson's disease, stereotactic stimulation at high radiofrequency(130 Hz) of the subthalamic nucleus (STN, corpus luysi) has shownefficacy in seizure control in the absence of apparent anatomicallesioning. In the latter case, the STN was not the focus per se and thestimulation protocol affected the network propagation of seizures ratherthan the source focus itself. Benabid A. L. et al., Deep brainstimulation of the corpus luysi (subthalamic nucleus) and other targetsin Parkinson's disease. Extension to new indications such as dystoniaand epilepsy, J Neurol 248(Suppl 3); 37-47: 2001.

Stereotactic radiosurgery (radiotherapy) is a noninvasive butirreversible procedure involving guided, selective irradiation of atargeted substructure, and has been described for the treatment ofseizures arising from a temporal focus. Cmelak A. J. et al., Low-dosestereotactic radiosurgery is inadequate for medically intractable mesialtemporal lobe epilepsy: a case report, Seizure 10; 442-446; 2001.Heikkinen E. R. et al., Stereotactic radiotherapy instead ofconventional epilepsy surgery. A case report, Acta Neurochir (Wien)119(1-4); 159-160: 1992. The effectiveness of radiotherapy isdose-dependent in both clinical (10 Gy, Heikkinen et al, 1992; 15 Gy,Cmelak et al, 2001) and experimental application (40 Gy, Sun B. et al.,Reduction of hippocampal-kindled seizure activity in rats bystereotactic radiosurgery, Exper Neurol 154; 691-695: 1998). Lower doseeffects are equivocal while higher doses are effective; however, higherdoses also produce histological, irreversible changes not seen at lowerdoses. Thus, anatomical lesioning of the target structure appears to beimportant in the efficacy of stereotactic radiosurgery, but mayintroduce dose-dependent toxicity issues.

Stereotactic procedures can also be used to guide the direct delivery ofpharmacologically active agents into specific, localized substructures.These agents may result in either permanent (irreversible) lesions, suchas with excitotoxins (e.g. kainic and ibotenic acids), or reversibleinactivation, such as with anesthetics and neurotransmitteriontophoresis. Lomber S. G., The advantages and limitations of permanentor reversible deactivation techniques in the assessment of neuralfunction, J Neurosci Meth 86; 109-117: 1999. Delivery may be achievedthrough both hypodermic cannulae and fine-tipped micropipettes, orvariations thereof. Malpeli J. G., Reversible inactivation ofsubcortical sites by drug injection, J Neurosci Meth 86; 119-128: 1999.Stereotactic placement of electrodes to record intracranial EEG has beendescribed together with simultaneous metabolic assessment throughmicrodialysis sampling of the target. Fried I. et al., Cerebralmicrodialysis combined with single-neuron and electroencephalographicrecording in neurosurgical patients, J Neurosurg 91; 697-705: 1999.Thus, it is possible, to configure a system to assess the neuronalactivity within the desired target before, during and after delivery ofthe pharmacological agent. With any such procedure, assessment of thespatial distribution (spread) of the agent within the target structurecan also be determined. Malpeli, 1999, supra; Martin J. H. et al.,Pharmacological inactivation in the analysis of the central control ofmovement, J Neurosci Meth 86; 145-159: 1999. Apart from neuronalactivity measured through indwelling depth electrodes, functional(metabolic) assessments can be made with PET imaging to determine theprofile and duration of effects of the active agent on the target inquestion.

In cases where subcortical structures encompass, or are in proximity to,critical functional areas, and thus cannot be approached with standardresective techniques, the treatment regimen may be a maintenance ofmultiple AEDs for suppression of seizure activity (Kwan and Brodie, 2002ibid). For functionally critical (eloquent) cortical regions that cannotbe approached with standard resective surgical procedures, multiplesubpial transections (MST) can be used to treat refractory seizures(Mulligan L. P. et al, Multiple subpial transections: the Yaleexperience. Epilepsia 42(2):226-229:2001). new approach to the surgicaltreatment of focal epilepsy, J Neurosurg 70: 231-239:1989) Thisprocedure exploits the natural orthogonal architecture of the cerebralcortex, which is arranged such that functional units are organizedvertically while intracortical interconnections are primarily horizontal(perpendicular) to the functional barrels. Synchronous epilepticactivity can arise through horizontal intracortical spread. The MSTprocedure physically disrupts these horizontal interconnections whilesparing the vertically-oriented cortical function (Morrell F. et al,Multiple subpial transection: a The MST procedure physically disruptsthese horizontal interconnections while sparing the vertically-orientedcortical function (Morrell et al, 1989 ibid). Thus, MST is a surgicaltechnique for intractable epilepsy which reduces the occurrence ofelectrocorticographical spikes recorded from the cortical surface andcan thereby reduce clinical epileptic events. Significantly, MST permitstreatment of unresectable epileptogenic foci, such as foci infunctionally important (“eloquent”) areas of the cerebral cortex.Resection of eloquent foci can leave an epilepsy patient with asignificant reduction in neurological function.

MST has as its' basis the diminution of the spread of electricalactivity between nearby superficial cortical neurons and is based uponthe anatomic and functional structure of the columnar organization ofthe higher mammalian neocortex. When used to effectively accomplish aseizure ocus resection, MST reduces the side to side spread ofepileptogenic energy along cortical neurons without causing majorfunctional impairment of the centrifugal and centripetal corticalneuronal connections. Thus, MST reduces the horizontal “cross talk” ofepileptic discharges in order to prevent the discharge spreading alongthe cortical service and the resulting seizure.

Brain Motor Systems

Several areas of the cerebrum influence motor activity. Thus, lesion tothe motor cortex of the cerebrum, as can result from stoke, can removeinhibition of vestibular and reticular brain stem nuclei, which thenbecome spontaneously active and cause spasm of muscles influenced by,the now disinhibited, lower brain areas.

An accessory motor system of the cerebrum is the basal ganglia. Thebasal ganglia receives most input from and sends most of its signalsback to the cortex. The basal ganglia include the caudate nucleus,putamen, globus pallidus, substantia nigra (which includes the parscompacta) and subthalamic nucleus. Because abnormal signals from thebasal ganglia to the motor cortex cause most of the abnormalities inParkinson's disease, attempts have been made to treat Parkinson'sdisease by blocking these signals. Thus lesions have been made in theventrolateral and ventroanterior nuclei of the thalamus to block thefeedback circuit from the basal ganglia to the cortex. Additionally,pallidotomy, the surgical ablation of part of the globus pallidus, hasbeen used to effectively treat the motor disorders of Parkinson'sdisease.

Surgical intervention is believed to assist by interrupting a motoricpathway which, due to a dopaminergic deficiency, had pathologicallyinhibited the thalamus. The inhibited thalamus in turn understimulatescortical neuronal networks responsible for generating movement. Hence,surgery removes the thalamic inhibition and has been used in thetreatment of pharmacoresistant movement disorders. Speelman, J. D., etal., Thalamic Surgery and Tremor, Mov Dis 13(3); 103-106:1998.

Intracranial lesions for the treatment of tremor and other parkinsoniansymptoms have been made to the globus pallidus and the ansalenticularis. Long term results of pallidotomy have sometimes beendisappointing. Positive results for the surgical arrest of tremor havebeen obtained by lesioning the following thalamic nuclei: (1) theventrointermedius (Vim) or ventral lateral posterior (VLp) nucleus; (2)ventrooralis anterior (Voa) nucleus (Voa and Vop have been collectivelytermed the ventral lateral anterior nucleus (VLa)); (3) ventrooralisposterior (Vop) nucleus; (4) subthalamic nuclei (campotomy), and; (5)CM-Pf thalamic nuclei. Generally, the ventrolateral thalamus has beenthe surgical target of choice in the treatment of Parkinson's diseaseand other systemically administered, drug resistant tremors. Brophy, B.P., et al., Thalamotomy for Parkinsonian Tremor, Stereotact FunctNeurosurg, 69; 1-4:1997. Thalamic excitation of the cortex is necessaryfor almost all cortical activity.

Stereotactic surgery (assisted by neuroimaging and electrophysiologicrecordings) has been used in the management of advanced,pharmacoresistant Parkinson's disease, targeting hyperactive globuspallidus and subthalamic nuclei. An electrode or a probe is placed intothe brain using a brain atlas for reference with assistance from brainimaging by computer tomography or magnetic resonance imaging. Lesions indifferent parts of the pallidum (i.e. posteroventral pallidum), basalganglia, thalamus and subthalamic nuclei have been carried out to treatmotor disorders of Parkinson's disease. Unfortunately, surgical brainlesions create a risk of impairment to speech, visual and cognitivebrain areas. Neurotransplantation shows promise but requires furtherinvestigation. Additionally, deep brain stimulation using electrodes forthe suppression of tremor using can create problems due to wire erosion,lead friction, infection of the implantable pulse generator, malfunctionof the implantable pulse generator, electrical shock and lead migration.Other complications due to electrode stimulation can include dysarthria,disequilibrium, paresis and gait disorder. See e.g. Koller, W. C. etal., Surgical Treatment of Parkinson's Disease, J Neurol Sci 167;1-10:1999, and Schuurman P. R., et al., A Comparison of ContinuousThalamic Stimulation and Thalamotomy for Suppression of Severe Tremor,NEJM 342(7); 461-468:2000.

Aside from surgical ablation or stimulation, external radiotherapy(Gamma Knife Radiosurgery) has also been used to a limited extent forthe treatment of drug resistant parkinsonian tremors. Drawbacks withthis procedure are that the reduction in tremor is delayed by betweenone week and eight months after the radiosurgery, and that long termbenefits as well as radiation side effects are currently unknown.

As set forth, treatment of parkinsonian tremor and other movementdisorders has been carried out by thalamotomy and/or interruption ofpallidofugal fibers in the subthalamic region and pallidotomy has alsobeen used. Current concepts of basal ganglia circuitry propose that theloss of striatal dopamine in Parkinson's disease leads to overactivityof the striatal projection to the lateral segment of the globuspallidus. The resulting decrease in lateral pallidal activity results indisinhibition of the subthalamic nucleus, its main projection site.Increased subthalamic activity in turn causes overactivity of theinternal segment of the globus pallidus, which projects to thepedunculopontine nucleus (PPN) and the ventrolateral (VL) thalamus.Thus, overactivity in the subthalamic nucleus and internal pallidumproduces the parkinsonian symptoms of tremor, bradykinesia andhypokinesia through projections to the PPN and VL thalamus. Lesion inthe subthalamic nucleus and the results of pallidotomy, particularlyposteroventral pallidotomy, have permitted effective treatment ofakinesia in parkinsonian patients.

Botulinum Toxin

The genus Clostridium has more than one hundred and twenty sevenspecies, grouped according to their morphology and functions. Theanaerobic, gram positive bacterium Clostridium botulinum produces apotent polypeptide neurotoxin, botulinum toxin, which causes aneuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

Botulinum toxin type A is the most lethal natural biological agent knownto man. About 50 picograms of a commercially available botulinum toxintype A (purified neurotoxin complex)¹ is a LD₅₀ in mice (i.e. 1 unit).One unit of BOTOX® contains about 50 picograms (about 56 attomoles) ofbotulinum toxin type A complex. Interestingly, on a molar basis,botulinum toxin type A is about 1.8 billion times more lethal thandiphtheria, about 600 million times more lethal than sodium cyanide,about 30 million times more lethal than cobra toxin and about 12 milliontimes more lethal than cholera. Singh, Critical Aspects of BacterialProtein Toxins, pages 63-84 (chapter 4) of Natural Toxins II, edited byB. R. Singh et al., Plenum Press, New York (1996) (where the stated LD₅₀of botulinum toxin type A of 0.3 ng equals 1 U is corrected for the factthat about 0.05 ng of BOTOX® equals 1 unit). One unit (U) of botulinumtoxin is defined as the LD₅₀ upon intraperitoneal injection into femaleSwiss Webster mice weighing 18 to 20 grams each. ¹Available fromAllergan, Inc., of Irvine, Calif. under the tradename BOTOX® in 100 unitvials

Seven immunologically distinct botulinum neurotoxins have beencharacterized, these being respectively botulinum neurotoxin serotypesA, B, C₁, D, E, F and G each of which is distinguished by neutralizationwith type-specific antibodies. The different serotypes of botulinumtoxin vary in the animal species that they affect and in the severityand duration of the paralysis they evoke. For example, it has beendetermined that botulinum toxin type A is 500 times more potent, asmeasured by the rate of paralysis produced in the rat, than is botulinumtoxin type B. Additionally, botulinum toxin type B has been determinedto be non-toxic in primates at a dose of 480 U/kg which is about 12times the primate LD₅₀ for botulinum toxin type A. Moyer E et al.,Botulinum Toxin Type B: Experimental and Clinical Experience, beingchapter 6, pages 71-85 of “Therapy With Botulinum Toxin”, edited byJankovic, J. et al. (1994), Marcel Dekker, Inc. Botulinum toxinapparently binds with high affinity to cholinergic motor neurons, istranslocated into the neuron and blocks the release of acetylcholine.

Regardless of serotype, the molecular mechanism of toxin intoxicationappears to be similar and to involve at least three steps or stages. Inthe first step of the process, the toxin binds to the presynapticmembrane of the target neuron through a specific interaction between theheavy chain, H chain, and a cell surface receptor; the receptor isthought to be different for each type of botulinum toxin and for tetanustoxin. The carboxyl end segment of the H chain, H_(C), appears to beimportant for targeting of the toxin to the cell surface.

In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This step is thought to be mediated by the amino end segment ofthe H chain, H_(N), which triggers a conformational change of the toxinin response to a pH of about 5.5 or lower. Endosomes are known topossess a proton pump which decreases intra-endosomal pH. Theconformational shift exposes hydrophobic residues in the toxin, whichpermits the toxin to embed itself in the endosomal membrane. The toxin(or at a minimum the light chain) then translocates through theendosomal membrane into the cytoplasm.

The last step of the mechanism of botulinum toxin activity appears toinvolve reduction of the disulfide bond joining the heavy chain, Hchain, and the light chain, L chain. The entire toxic activity ofbotulinum and tetanus toxins is contained in the L chain of theholotoxin; the L chain is a zinc (Zn++) endopeptidase which selectivelycleaves proteins essential for recognition and docking ofneurotransmitter-containing vesicles with the cytoplasmic surface of theplasma membrane, and fusion of the vesicles with the plasma membrane.Tetanus neurotoxin, botulinum toxin types B, D, F, and G causedegradation of synaptobrevin (also called vesicle-associated membraneprotein (VAMP)), a synaptosomal membrane protein. Most of the VAMPpresent at the cytoplasmic surface of the synaptic vesicle is removed asa result of any one of these cleavage events. Botulinum toxin serotype Aand E cleave SNAP-25. Botulinum toxin serotype C₁ was originally thoughtto cleave syntaxin, but was found to cleave syntaxin and SNAP-25. Eachof the botulinum toxins specifically cleaves a different bond, exceptbotulinum toxin type B (and tetanus toxin) which cleave the same bond.

Although all the botulinum toxins serotypes apparently inhibit releaseof the neurotransmitter acetylcholine at the neuromuscular junction,they do so by affecting different neurosecretory proteins and/orcleaving these proteins at different sites. For example, botulinum typesA and E both cleave the 25 kiloDalton (kD) synaptosomal associatedprotein (SNAP-25), but they target different amino acid sequences withinthis protein. Botulinum toxin types B, D, F and G act onvesicle-associated protein (VAMP, also called synaptobrevin), with eachserotype cleaving the protein at a different site. Finally, botulinumtoxin type C₁ has been shown to cleave both syntaxin and SNAP-25. Thesedifferences in mechanism of action may affect the relative potencyand/or duration of action of the various botulinum toxin serotypes.Apparently, a substrate for a botulinum toxin can be found in a varietyof different cell types. See e.g. Gonelle-Gispert, C., et al., SNAP-25aand -25b isoforms are both expressed in insulin-secreting cells and canfunction in insulin secretion, Biochem J. 1; 339 (pt 1): 159-65:1999,and Boyd R. S. et al., The effect of botulinum neurotoxin-B on insulinrelease from a ∃-cell line, and Boyd R. S. et al., The insulin secreting∃-cell line, HIT-15, contains SNAP-25 which is a target for botulinumneurotoxin-A, both published at Mov Disord, 10(3):376:1995 (pancreaticislet B cells contains at least SNAP-25 and synaptobrevin).

The molecular weight of the botulinum toxin protein molecule, for allseven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the botulinumtoxin type A complex can be produced by Clostridial bacterium as 900 kD,500 kD and 300 kD forms. Botulinum toxin types B and C₁ is apparentlyproduced as only a 700 kD or 500 kD complex. Botulinum toxin type D isproduced as both 300 kD and 500 kD complexes. Finally, botulinum toxintypes E and F are produced as only approximately 300 kD complexes. Thecomplexes (i.e. molecular weight greater than about 150 kD) are believedto contain a non-toxin hemaglutinin protein and a non-toxin andnon-toxic nonhemaglutinin protein. These two non-toxin proteins (whichalong with the botulinum toxin molecule comprise the relevant neurotoxincomplex) may act to provide stability against denaturation to thebotulinum toxin molecule and protection against digestive acids whentoxin is ingested. Additionally, it is possible that the larger (greaterthan about 150 kD molecular weight) botulinum toxin complexes may resultin a slower rate of diffusion of the botulinum toxin away from a site ofintramuscular injection of a botulinum toxin complex.

All the botulinum toxin serotypes are made by Clostridium botulinumbacteria as inactive single chain proteins which must be cleaved ornicked by proteases to become neuroactive. The bacterial strains thatmake botulinum toxin serotypes A and G possess endogenous proteases andserotypes A and G can therefore be recovered from bacterial cultures inpredominantly their active form. In contrast, botulinum toxin serotypesC₁, D, and E are synthesized by nonproteolytic strains and are thereforetypically unactivated when recovered from culture. Serotypes B and F areproduced by both proteolytic and nonproteolytic strains and thereforecan be recovered in either the active or inactive form. However, eventhe proteolytic strains that produce, for example, the botulinum toxintype B serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for a lowerpotency of botulinum toxin type B as compared to botulinum toxin type A.The presence of inactive botulinum toxin molecules in a clinicalpreparation will contribute to the overall protein load of thepreparation, which has been linked to increased antigenicity, withoutcontributing to its clinical efficacy.

Botulinum toxins and toxin complexes can be obtained from, for example,List Biological Laboratories, Inc., Campbell, Calif.; the Centre forApplied Microbiology and Research, Porton Down, U.K.; Wako (Osaka,Japan), as well as from Sigma Chemicals of St Louis, Mo. Commerciallyavailable botulinum toxin containing pharmaceutical compositions includeBOTOX® (Botulinum toxin type A neurotoxin complex with human serumalbumin and sodium chloride) available from Allergan, Inc., of Irvine,Calif. in 100 unit vials as a lyophilized powder to be reconstitutedwith 0.9% sodium chloride before use), Dysport® (Clostridium botulinumtype A toxin haemagglutinin complex with human serum albumin and lactosein the formulation), available from Ipsen Limited, Berkshire, U.K. as apowder to be reconstituted with 0.9% sodium chloride before use), andMyoBloc™ (an injectable solution comprising botulinum toxin type B,human serum albumin, sodium succinate, and sodium chloride at about pH5.6, available from Elan Corporation, Dublin, Ireland).

The success of botulinum toxin type A to treat a variety of clinicalconditions has led to interest in other botulinum toxin serotypes.Additionally, pure botulinum toxin has been used to treat humans. seee.g. Kohl A., et al., Comparison of the effect of botulinum toxin A(Botox®) with the highly-purified neurotoxin (NT 201) in the extensordigitorum brevis muscle test, Mov Disord 2000; 15(Suppl 3):165. Hence, apharmaceutical composition can be prepared using a pure botulinum toxin.

The type A botulinum toxin is known to be soluble in dilute aqueoussolutions at pH 4-6.8. At pH above about 7 the stabilizing nontoxicproteins dissociate from the neurotoxin, resulting in a gradual loss oftoxicity, particularly as the pH and temperature rise. Schantz E. J., etal Preparation and characterization of botulinum toxin type A for humantreatment (in particular pages 44-45), being chapter 3 of Jankovic, J.,et al, Therapy with Botulinum Toxin, Marcel Dekker, Inc (1994).

The botulinum toxin molecule (about 150 kDa), as well as the botulinumtoxin complexes (about 300-900 kDa), such as the toxin type A complexare also extremely susceptible to denaturation due to surfacedenaturation, heat, and alkaline conditions. Inactivated toxin formstoxoid proteins which may be immunogenic. The resulting antibodies canrender a patient refractory to toxin injection.

In vitro studies have indicated that botulinum toxin inhibits potassiumcation induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brainstem tissue. Additionally, it has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine (Habermann E., et al., Tetanus Toxin and Botulinum A andC Neurotoxins Inhibit Noradrenaline Release From Cultured Mouse Brain, JNeurochem 51(2);522-527:1988) CGRP, substance P and glutamate(Sanchez-Prieto, J., et al., Botulinum Toxin A Blocks GlutamateExocytosis From Guinea Pig Cerebral Cortical Synaptosomes, Eur J.Biochem 165; 675-681:1987. Thus, when adequate concentrations are used,stimulus-evoked release of most neurotransmitters is blocked bybotulinum toxin. See e.g. Pearce, L. B., Pharmacologic Characterizationof Botulinum Toxin For Basic Science and Medicine, Toxicon 35(9);1373-1412 at 1393; Bigalke H., et al., Botulinum A Neurotoxin InhibitsNon-Cholinergic Synaptic Transmission in Mouse Spinal Cord Neurons inCulture, Brain Research 360; 318-324:1985; Habermann E., Inhibition byTetanus and Botulinum A Toxin of the release of [ ³ H]Noradrenaline and[ ³ H]GABA From Rat Brain Homogenate, Experientia 44; 224-226:1988,Bigalke H., et al., Tetanus Toxin and Botulinum A Toxin Inhibit Releaseand Uptake of Various Transmitters, as Studied with ParticulatePreparations From Rat Brain and Spinal Cord, Naunyn-Schmiedeberg's ArchPharmacol 316; 244-251:1981, and; Jankovic J. et al., Therapy WithBotulinum Toxin, Marcel Dekker, Inc., (1994), page 5.

Botulinum toxin type A can be obtained by establishing and growingcultures of Clostridium botulinum in a fermenter and then harvesting andpurifying the fermented mixture in accordance with known procedures. Allthe botulinum toxin serotypes are initially synthesized as inactivesingle chain proteins which must be cleaved or nicked by proteases tobecome neuroactive. The bacterial strains that make botulinum toxinserotypes A and G possess endogenous proteases and serotypes A and G cantherefore be recovered from bacterial cultures in predominantly theiractive form. In contrast, botulinum toxin serotypes C₁, D and E aresynthesized by nonproteolytic strains and are therefore typicallyunactivated when recovered from culture. Serotypes B and F are producedby both proteolytic and nonproteolytic strains and therefore can berecovered in either the active or inactive form. However, even theproteolytic strains that produce, for example, the botulinum toxin typeB serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B as compared tobotulinum toxin type A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that botulinum toxin type B has, uponintramuscular injection, a shorter duration of activity and is also lesspotent than botulinum toxin type A at the same dose level.

High quality crystalline botulinum toxin type A can be produced from theHall A strain of Clostridium botulinum with characteristics of ≧3×10⁷U/mg, an A₂₆₀/A₂₇₈ of less than 0.60 and a distinct pattern of bandingon gel electrophoresis. The known Schantz process can be used to obtaincrystalline botulinum toxin type A, as set forth in Schantz, E. J., etal, Properties and use of Botulinum toxin and Other MicrobialNeurotoxins in Medicine, Microbiol Rev. 56; 80-99:1992. Generally, thebotulinum toxin type A complex can be isolated and purified from ananaerobic fermentation by cultivating Clostridium botulinum type A in asuitable medium. The known process can also be used, upon separation outof the non-toxin proteins, to obtain pure botulinum toxins, such as forexample: purified botulinum toxin type A with an approximately 150 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater; purified botulinum toxin type B with an approximately 156 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater, and; purified botulinum toxin type F with an approximately 155kD molecular weight with a specific potency of 1-2×10⁷ LD₅₀ U/mg orgreater.

Either the pure botulinum toxin (i.e. the 150 kilodalton botulinum toxinmolecule) or the toxin complex can be used to prepare a pharmaceuticalcomposition. Both molecule and complex are susceptible to denaturationdue to surface denaturation, heat, and alkaline conditions. Inactivatedtoxin forms toxoid proteins which may be immunogenic. The resultingantibodies can render a patient refractory to toxin injection.

As with enzymes generally, the biological activities of the botulinumtoxins (which are intracellular peptidases) is dependant, at least inpart, upon their three dimensional conformation. Thus, botulinum toxintype A is detoxified by heat, various chemicals surface stretching andsurface drying. Additionally, it is known that dilution of the toxincomplex obtained by the known culturing, fermentation and purificationto the much, much lower toxin concentrations used for pharmaceuticalcomposition formulation results in rapid detoxification of the toxinunless a suitable stabilizing agent is present. Dilution of the toxinfrom milligram quantities to a solution containing nanograms permilliliter presents significant difficulties because of the rapid lossof specific toxicity upon such great dilution. Since the toxin may beused months or years after the toxin containing pharmaceuticalcomposition is formulated, the toxin can stabilized with a stabilizingagent such as albumin and gelatin.

A commercially available botulinum toxin containing pharmaceuticalcomposition is sold under the trademark BOTOX® (available from Allergan,Inc., of Irvine, Calif.). BOTOX® consists of a purified botulinum toxintype A complex, albumin and sodium chloride packaged in sterile,vacuum-dried form. The botulinum toxin type A is made from a culture ofthe Hall strain of Clostridium botulinum grown in a medium containingN-Z amine and yeast extract. The botulinum toxin type A complex ispurified from the culture solution by a series of acid precipitations toa crystalline complex consisting of the active high molecular weighttoxin protein and an associated hemagglutinin protein. The crystallinecomplex is re-dissolved in a solution containing saline and albumin andsterile filtered (0.2 microns) prior to vacuum-drying. The vacuum-driedproduct is stored in a freezer at or below −5° C. BOTOX® can bereconstituted with sterile, non-preserved saline prior to intramuscularinjection. Each vial of BOTOX® contains about 100 units (U) ofClostridium botulinum toxin type A purified neurotoxin complex, 0.5milligrams of human serum albumin and 0.9 milligrams of sodium chloridein a sterile, vacuum-dried form without a preservative.

To reconstitute vacuum-dried BOTOX®, sterile normal saline without apreservative; (0.9% Sodium Chloride Injection) is used by drawing up theproper amount of diluent in the appropriate size syringe. Since BOTOX®may be denatured by bubbling or similar violent agitation, the diluentis gently injected into the vial. For sterility reasons BOTOX® ispreferably administered within four hours after the vial is removed fromthe freezer and reconstituted. During these four hours, reconstitutedBOTOX® can be stored in a refrigerator at about 2° C. to about 8° C.Reconstituted, refrigerated BOTOX® has been reported to retain itspotency for at least about two weeks. Neurology, 48:249-53:1997.

Botulinum toxins have been used in clinical settings for the treatmentof neuromuscular disorders characterized by hyperactive skeletalmuscles. Botulinum toxin type A (Botox®) was approved by the U.S. Foodand Drug Administration in 1989 for the treatment of essentialblepharospasm, strabismus and hemifacial spasm in patients over the ageof twelve. In 2000 the FDA approved commercial preparations of type A(Botox®) and type B botulinum toxin (MyoBloc™) serotypes for thetreatment of cervical dystonia, and in 2002 the FDA approved a type Abotulinum toxin (Botox®) for the cosmetic treatment of certainhyperkinetic (glabellar) facial wrinkles. Clinical effects of peripheralintramuscular botulinum toxin type A are usually seen within one week ofinjection and sometimes within a few hours. The typical duration ofsymptomatic relief (i.e. flaccid muscle paralysis) from a singleintramuscular injection of botulinum toxin type A can be about threemonths, although in some cases the effects of a botulinum toxin induceddenervation of a gland, such as a salivary gland, have been reported tolast for several years. For example, it is known that botulinum toxintype A can have an efficacy for up to 12 months (Naumann M., et al.,Botulinum toxin type A in the treatment of focal, axillary and palmarhyperhidrosis and other hyperhidrotic conditions, European J. Neurology6 (Supp 4): S111-S115:1999), and in some circumstances for as long as 27months. Ragona, R. M., et al., Management of parotid sialocele withbotulinum toxin, The Laryngoscope 109:1344-1346:1999. However, the usualduration of an intramuscular injection of Botox® is typically about 3 to4 months.

It has been reported that a botulinum toxin type A has been used indiverse clinical settings, including for example as follows:

(1) about 75-125 units of BOTOX® per intramuscular injection (multiplemuscles) to treat cervical dystonia;

(2) 5-10 units of BOTOX® per intramuscular injection to treat glabellarlines (brow furrows) (5 units injected intramuscularly into the procerusmuscle and 10 units injected intramuscularly into each corrugatorsupercilii muscle);

(3) about 30-80 units of BOTOX® to treat constipation by intrasphincterinjection of the puborectalis muscle;

(4) about 1-5 units per muscle of intramuscularly injected BOTOX® totreat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.

(5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of dioptercorrection desired).

(6) to treat upper limb spasticity following stroke by intramuscularinjections of BOTOX® into five different upper limb flexor muscles, asfollows:

-   -   (a) flexor digitorum profundus: 7.5 U to 30 U    -   (b) flexor digitorum sublimus: 7.5 U to 30 U    -   (c) flexor carpi ulnaris: 10 U to 40 U    -   (d) flexor carpi radialis: 15 U to 60 U    -   (e) biceps brachii: 50 U to 200 U. Each of the five indicated        muscles has been injected at the same treatment session, so that        the patient receives from 90 U to 360 U of upper limb flexor        muscle BOTOX® by intramuscular injection at each treatment        session.

(7) to treat migraine, pericranial injected (injected symmetrically intoglabellar, frontalis and temporalis muscles) injection of 25 U of BOTOX®has showed significant benefit as a prophylactic treatment of migrainecompared to vehicle as measured by decreased measures of migrainefrequency, maximal severity, associated vomiting and acute medicationuse over the three month period following the 25 U injection.

Additionally, intramuscular botulinum toxin has been used in thetreatment of tremor in patients with Parkinson's disease, although ithas been reported that results have not been impressive. Marjama-Lyons,J., et al., Tremor-Predominant Parkinson's Disease, Drugs & Aging 16(4);273-278:2000.

Treatment of certain gastrointestinal and smooth muscle disorders with abotulinum toxin are known. See e.g. U.S. Pat. Nos. 5,427,291 and5,674,205 (Pasricha). Additionally, transurethral injection of abotulinum toxin into a bladder sphincter to treat a urination disorderis known (see e.g. Dykstra, D. D., et al, Treatment ofdetrusor-sphincter dyssynergia with botulinum A toxin: A double-blindstudy, Arch Phys Med Rehabil 1990 January; 71:24-6), as is injection ofa botulinum toxin into the prostate to treat prostatic hyperplasia. Seee.g. U.S. Pat. No. 6,365,164 (Schmidt).

U.S. Pat. No. 5,766,605 (Sanders) proposes the treatment of variousautonomic disorders, such as hypersalivation and rhinittis, with abotulinum toxin.

Furthermore, various afflictions, such as hyperhydrosis and headache,treatable with a botulinum toxin are discussed in WO 95/17904(PCT/US94/14717) (Aoki). EP 0 605 501 B1 (Graham) discusses treatment ofcerebral palsy with a botulinum toxin and U.S. Pat. No. 6,063,768(First) discusses treatment of neurogenic inflammation with a botulinumtoxin.

Botulinum toxin has been used to study the release of dopamine fromvarious brain cells, in light of the theory that Parkinson's disease isdue to the death of dopamine releasing cell in the striatal region ofthe brain. See e.g. Bergquist F. et al., Evidence for differentexocytosis pathways in dendritic and terminal dopamine release in vivo,Brain Research 950; 245-253: (2002), which me determined that in vivo,intracranial microdialysis of a botulinum toxin type A into either thesubstantia nigra or striatum use of rats strongly reduced release ofsomatodentritic dopamine from cells therein. Hence, one could concludethat evidence that the death of dopamine releasing cells in either thesubstantia nigra or substantia striatum regions of the brain can be afactor in the occurrence of Parkinson's disease.

In addition to having pharmacologic actions at the peripheral location,botulinum toxins can also have inhibitory effects in the central nervoussystem. Work by Weigand et al, (¹²⁵ I-labelled botulinum Aneurotoxin:pharmacokinetics in cats after intramuscular injection,Nauny-Schmiedeberg's Arch. Pharmacol. 1976; 292, 161-165), andHabermann, (¹²⁵ I-labelled Neurotoxin from clostridium botulinum A:preparation, binding to synaptosomes and ascent to the spinal cord,Nauny-Schmiedeberg's Arch. Pharmacol. 1974; 281, 47-56) showed thatbotulinum toxin is able to ascend to the spinal area by retrogradetransport. As such, a botulinum toxin injected at a peripheral location,for example intramuscularly, may be retrograde transported to the spinalcord.

In vitro studies have indicated that botulinum toxin inhibits potassiumcation induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brainstem tissue. Additionally, it has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine, CGRP and glutamate.

U.S. Pat. No. 5,989,545 discloses that a modified clostridial neurotoxinor fragment thereof, preferably a botulinum toxin, chemically conjugatedor recombinantly fused to a particular targeting moiety can be used totreat pain by administration of the agent to the spinal cord.

Tetanus toxin, as wells as derivatives (i.e. with a non-native targetingmoiety), fragments, hybrids and chimeras thereof can also havetherapeutic utility. The tetanus toxin bears many similarities to thebotulinum toxins. Thus, both the tetanus toxin and the botulinum toxinsare polypeptides made by closely related species of Clostridium(Clostridium tetani and Clostridium botulinum, respectively).Additionally, both the tetanus toxin and the botulinum toxins aredichain proteins composed of a light chain (molecular weight about 50kD) covalently bound by a single disulfide bond to a heavy chain(molecular weight about 100 kD). Hence, the molecular weight of tetanustoxin and of each of the seven botulinum toxins (non-complexed) is about150 kD. Furthermore, for both the tetanus toxin and the botulinumtoxins, the light chain bears the domain which exhibits intracellularbiological (protease) activity, while the heavy chain comprises thereceptor binding (immunogenic) and cell membrane translocationaldomains.

Further, both the tetanus toxin and the botulinum toxins exhibit a high,specific affinity for gangliocide receptors on the surface ofpresynaptic cholinergic neurons. Receptor mediated endocytosis oftetanus toxin by peripheral cholinergic neurons results in retrogradeaxonal transport, blocking of the release of inhibitoryneurotransmitters from central synapses and a spastic paralysis.Contrarily, receptor mediated endocytosis of botulinum toxin byperipheral cholinergic neurons results in little if any retrogradetransport, inhibition of acetylcholine exocytosis from the intoxicatedperipheral motor neurons and a flaccid paralysis.

The tetanus toxin and the botulinum toxins resemble each other in bothbiosynthesis and molecular architecture. Thus, there is an overall 34%identity between the protein sequences of tetanus toxin and botulinumtoxin type A, and a sequence identity as high as 62% for some functionaldomains. Binz T. et al., The Complete Sequence of Botulinum NeurotoxinType A and Comparison with Other Clostridial Neurotoxins, J BiologicalChemistry 265(16); 9153-9158:1990.

It is known that injection of tetanus toxin into rat cerebral cortex caninduce chronic epilepsy and based upon this finding tetanus toxin hasbeen used to provide a model of focal epilepsies. See e.g. Mellanby, J.,Tetanus toxin as a tool for investigating the consequences of excessiveneuronal excitation, pages 291-297 of Dasgupta, B. R., Botulinum andtetanus neurotoxins, Plenum Press (1993), and; Jefferys, J. G., et al.,Chronic focal epilepsy induced by intracerebral tetanus toxin, Ital JNeurol Sci 1995 February-March; 16(1-2):27-32.

Contrary to the result upon intracranial administration of a tetanustoxin, seizures do not result upon intrathecal administration of abotulinum toxin to a mammal (see e.g. U.S. Pat. No. 6,113,915).Additionally, and as set forth above, intoxication with tetanus toxinresults in a spastic paralysis, whereas intoxication with a botulinumtoxin results in a flaccid paralysis. Intramuscular administration of abotulinum toxin to treat epilepsy has been reported to be ineffective.Tarsy, D. et al., Botulinum toxin treatment is not effective forepilepsy partialis continua, Mov Disord 1995; 10(3):357-8.

Additionally, intraventricular injection of tetanus toxin can cause anincrease in serotonin levels in rats. Aguilera, J. et al., Stereotaxicinjection of tetanus toxin in rat central nervous system causesalteration in normal levels of monoamines, J. Neurochem, 56(3); 733-738:1991.

Acetylcholine

Typically only a single type of small molecule neurotransmitter isreleased by each type of neuron in the mammalian nervous system. Theneurotransmitter acetylcholine is secreted by neurons in many areas ofthe brain, but specifically by the large pyramidal cells of the motorcortex, by several different neurons in the basal ganglia, by the motorneurons that innervate the skeletal muscles, by the preganglionicneurons of the autonomic nervous system (both sympathetic andparasympathetic), by the postganglionic neurons of the parasympatheticnervous system, and by some of the postganglionic neurons of thesympathetic nervous system. Essentially, only the postganglionicsympathetic nerve fibers to the sweat glands, the piloerector musclesand a few blood vessels are cholinergic, as most of the postganglionicneurons of the sympathetic nervous system secret the neurotransmitternorepinephrine. In most instances acetylcholine has an excitatoryeffect. However, acetylcholine is known to have inhibitory effects atsome of the peripheral parasympathetic nerve endings, such as inhibitionof heart rate by the vagal nerve.

The efferent signals of the autonomic nervous system are transmitted tothe body through either the sympathetic nervous system or theparasympathetic nervous system. The preganglionic neurons of thesympathetic nervous system extend from preganglionic sympathetic neuroncell bodies located in the intermediolateral horn of the spinal cord.The preganglionic sympathetic nerve fibers, extending from the cellbody, synapse with postganglionic neurons located in either aparavertebral sympathetic ganglion or in a prevertebral ganglion. Since,the preganglionic neurons of both the sympathetic and parasympatheticnervous system are cholinergic, application of acetylcholine to theganglia will excite both sympathetic and parasympathetic postganglionicneurons.

Acetylcholine activates two types of receptors, muscarinic and nicotinicreceptors. The muscarinic receptors are found in all effector cellsstimulated by the postganglionic, neurons of the parasympathetic nervoussystem as well as in those stimulated by the postganglionic cholinergicneurons of the sympathetic nervous system. The nicotinic receptors arefound in the adrenal medulla, as well as within the autonomic ganglia,that is on the cell surface of the postganglionic neuron at the synapsebetween the preganglionic and postganglionic neurons of both thesympathetic and parasympathetic systems. Nicotinic receptors are alsofound in many nonautonomic nerve endings, for example in the membranesof skeletal muscle fibers at the neuromuscular junction.

Acetylcholine is released from cholinergic neurons when small, clear,intracellular vesicles fuse with the presynaptic neuronal cell membrane.A wide variety of non-neuronal secretory cells, such as, adrenal medulla(as well as the PC12 cell line) and pancreatic islet cells releasecatecholamines and parathyroid hormone, respectively, from largedense-core vesicles. The PC 2 cell line is a clone of ratpheochromocytoma cells extensively used as a tissue culture model forstudies of sympathoadrenal development. Botulinum toxin inhibits therelease of both types of compounds from both types of cells in vitro,permeabilized (as by electroporation) or by direct injection of thetoxin into the denervated cell. Botulinum toxin is also known to blockrelease of the neurotransmitter glutamate from cortical synaptosomescell cultures.

A neuromuscular junction is formed in skeletal muscle by the proximityof axons to muscle cells. A signal transmitted through the nervoussystem results in an action potential at the terminal axon, withactivation of ion channels and resulting release of the neurotransmitteracetylcholine from intraneuronal synaptic vesicles, for example at themotor endplate of the neuromuscularjunction. The acetylcholine crossesthe extracellular space to bind with acetylcholine receptor proteins onthe surface of the muscle end plate. Once sufficient binding hasoccurred, an action potential of the muscle cell causes specificmembrane ion channel changes, resulting in muscle cell contraction. Theacetylcholine is then released from the muscle cells and metabolized bycholinesterases in the extracellular space. The metabolites are recycledback into the terminal axon for reprocessing into further acetylcholine.

Cholinergic Brain Systems

Cholinergic influence of both the motor and visual thalamus originatesfrom both the brainstem and the basal forebrain. See e.g. Billet S., etal., Cholinergic Projections to the Visual Thalamus and SuperiorColliculus, Brain Res. 847; 121-123:1999 and Oakman, S. A. et al.,Characterization of the Extent of Pontomesencephalic CholinergicNeurons' projections to the Thalamus: Comparison with Projections toMidbrain Dopaminergic Groups, Neurosci 94(2);529-547; 1999. Thus, it isknown based on histochemical studies using acetylcholinesterase (AchE)staining and retrograde tracing with choline acetyltransferase (ChAT)immunochemistry that there can be ascending cholinergic stimulation bythe brainstem of thalamic neurons. Steriade M. et al., Brain CholinergicSystems, Oxford University Press (1990), chapter 1. Indeed, manythalamic nuclei receive dense cholinergic innervation from brainstemreticular formations. Ibid, page 167. Known brainstem cholinergic cellgroups are located within: (1) the rostral pons at what is termed a Ch5location, which is located within the central tegmental field around thebrachium conjunctivum, forming a pedunculopontine tegmental nucleus,and; (2) the caudal part of the midbrain, at what is termed a Ch6location, the laterodorsal tegmental nucleus, which is embedded in theperiaqueductal and periventricular gray matter. The Ch5 and Ch6 cellgroups can consist almost exclusively of cholinergic neurons andtogether form the pontine cholinergic system. The Ch5-Ch6 cholinergicgroups provide direct ascending projections that terminate in a numberof target structure in the midbrain, diencephalon and telencephalon,including the superior colliculus, anterior pretectal area, interstitialmagnocellular nucleus of the posterior commissure, lateral habenularnucleus, thalamus, magnocellular preoptic nucleus, lateral mammillarynucleus, basal forebrain, olfactory bulb, medial prefrontal cortex andpontine nuclei. Stone T. W., CNS Neurotransmitters and Neuromodulators:Acetylcholine, CRC Press (1995), page 16. See also Schafer M. K.-H. etal., Cholinergic Neurons and Terminal Fields Revealed by Immunochemistryfor the Vesicular Acetylcholine Transporter. I. Central Nervous System,Neuroscience, 84(2); 331-359:1998. Three dimensional localization ofCh1-8 cholinergic nuclei have been mapped in humans. See e.g. Tracey, D.J., et al., Neurotransmitters in the Human Brain, Plenum Press (1995),pages 136-139.

Additionally, the basal forebrain (proencephalon) provides cholinergicinnervation of the dorsal thalamus, as well as to the neocortex,hippocampus, amygdala and olfactory bulb. See e.g. Steridae, page136-136, supra. Basal forebrain areas where the great proportion ofneurons are cholinergic include the medial septal nucleus (Ch1), thevertical branches of the diagonal band nuclei (Ch2), the horizontalbranches of the diagonal band nuclei (Ch3), and the magnocellularnucleus basalis (Ch4), which is located dorsolaterally to the Ch3 cellgroup. Ch1 and Ch2 provide the major component of cholinergic projectionto the hippocampus. The cells in the Ch3 sector project to the olfactorybulb.

Furthermore, cholinergic neurons are present in the thalamus. Rico, B.et al., A Population of Cholinergic Neurons is Present in the MacaqueMonkey Thalamus, Eur J Neurosci, 10; 2346-2352:1998.

Abnormalities in the brain's cholinergic system have been consistentlyidentified in a variety of neuropsychiatric disorders includingAlzheimer's disease, Parkinson's disease and dementia with Lewy bodies.Thus, in Alzheimer's disease there is hypoactivity of cholinergicprojections to the hippocampus and cortex. In individuals with dementiawith Lewy bodies extensive neocortical cholinergic deficits are believedto exist and in Parkinson's disease there is a loss of pedunculopontinecholinergic neurons. Notably, in vivo imaging of cholinergic activity inthe human brain has been reported. Perry, et al., Acetylcholine in Mind:a Neurotransmitter Correlate of Consciousness?, TINS 22(6); 273-280:1999

As set forth, current therapies relating to neuronal inhibition, such asresection. radiosurgery ablation, microinjection of pharmacologicalagents confirm that suppression of an epileptogenic focus can arrestseizures. Unfortunately such methods of treating focal epilepsy eitherprovide transient effects, irreversible damage to brain tissue orintroduce unwanted toxicity.

What is needed therefore is an intracranial method for effectivelytreating focal epilepsies by administration of a pharmaceutical whichhas the characteristics of long duration of activity, low rates ofdiffusion out of a chosen intracranial target tissue where administered,and nominal systemic effects at therapeutic dose levels.

SUMMARY

The present invention meets this need and provides methods foreffectively treating focal epilepsies by intracranial administration ofa Clostridial neurotoxin which has the characteristics of long durationof activity, low rates of diffusion out of an intracranial site whereadministered and insignificant systemic effects at therapeutic doselevels.

The following definitions apply herein:

“About” means approximately or nearly and in the context of a numericalvalue or range set forth herein means ±10% of the numerical value orrange recited or claimed.

“Biological activity” includes, with regard to a neurotoxin, the abilityto influence synthesis, exocytosis, receptor binding and/or uptake of aneurotransmitter, such as acetylcholine, or of an endocrine or exocrinesecretory product, such as insulin or pancreatic juice, respectively.

“Local administration” means direct administration of a pharmaceuticalat or to the vicinity of a site on or within a patients' body, at whichsite a biological effect of the pharmaceutical is desired. Localadministration excludes systemic routes of administration, such asintravenous or oral administration.

“Neurotoxin” means a biologically active molecule with a specificaffinity for a neuronal cell surface receptor. Neurotoxin includesClostridial toxins both as pure toxin and as complexed with one to morenon-toxin, toxin associated proteins

“Intracranial” means within the cranium or at or near the dorsal end ofthe spinal cord and includes the medulla, brain stem, pons, cerebellumand cerebrum.

“Intraparenchymal” means within the parenchyma of the brain, that iswithin the tissue (including within an extracellular space of braintissue) of the brain, as opposed to being within a ventricle of thebrain.

A method within the scope of the present invention can be used to treatepilepsy, including: (1) focal (or partial) epilepsies, such as, benignoccipital epilepsy (benign focal epilepsy with occipital paroxysms),benign rolandic epilepsy (benign focal epilepsy with centrotemporalspikes), frontal lobe epilepsy, occipital lobe epilepsy, mesial temporallobe epilepsy and parietal lobe epilepsy; (2) generalized idiopathicepilepsies, such as benign myoclonic epilepsy in infants, juvenilemyoclonic epilepsy, childhood absence epilepsy, juvenile absenceepilepsy, and epilepsy with generalized tonic clonic seizures inchildhood; (3) generalized symptomatic epilepsies, such as infantilespasms (West syndrome), Lennox-Gastaut syndrome and progressivemyoclonus epilepsies, and; (4) unclassified epilepsies, such as febrilefits, epilepsy with continuous spike and waves in slow wave sleep(ESES), Landau Kleffner syndrome, Rasmussen's syndrome and epilepsy andinborn errors in metabolism

A method for treating a movement disorder within the scope of thepresent invention can be by intracranial administration of a neurotoxinto a patient to thereby alleviate a symptom of the movement disorder.The neurotoxin is made by a bacterium selected from the group consistingof Clostridium botulinum, Clostridium butyricum and Clostridium beratti,or can be expressed by a suitable host (i.e. a recombinantly altered E.coli) which encodes for a neurotoxin made by Clostridium botulinum,Clostridium butyricum or Clostridium beratti. Preferably, the neurotoxinis a botulinum toxin, such as a botulinum toxin type A, B, C₁, D, E, Fand G.

The neurotoxin can be administered to various brain areas fortherapeutic treatment of a movement disorder, including to a lower brainregion, to a pontine region, to a mesopontine region, to a globuspallidus and/or to a thalamic region of the brain.

The neurotoxin can be a modified neurotoxin, that is a neurotoxin whichhas at least one of its amino acids deleted, modified or replaced, ascompared to a native or the modified neurotoxin can be a recombinantproduced neurotoxin or a derivative or fragment thereof.

Intracranial administration of a neurotoxin according to the presentinvention can include the step of implantation of controlled releasebotulinum toxin system. A detailed embodiment of the present inventioncan be a method for treating a movement disorder by intracranialadministration of a therapeutically effective amount of a botulinumtoxin to a patient to thereby treating a symptom of a movement disorder.The movement disorders treated can include Parkinson's disease,Huntington's Chorea, progressive supranuclear palsy, Wilson's disease,Tourette's syndrome, epilepsy, chronic tremor, tics, dystonias andspasticity

A further embodiment within the scope of the present invention can be amethod for treating epilepsy, the method comprising the steps of:selecting a neurotoxin with tremor suppressant activity; choosing anintracranial target tissue which influences a movement disorder; and;intracranially administering to the target tissue a therapeuticallyeffective amount of the neurotoxin selected, thereby treating theepilepsy.

Thus, a method for treating an epilepsy according to the presentinvention can have the step of intracranial administration of aneurotoxin to a mammal, thereby alleviating a symptom of an epilepsyexperienced by the mammal. Most preferably, the botulinum toxin used isbotulinum toxin type A because of the high potency, ready availabilityand long history of clinical use of botulinum toxin type A to treatvarious disorders.

We have surprisingly found that a botulinum toxin, such as botulinumtoxin type A, can be intracranially administered in amounts betweenabout 10⁻³ U/kg and about 10 U/kg to alleviate a focal epilepsy disorderexperienced by a human patient. Preferably, the botulinum toxin used isintracranially administered in an amount of between about 10⁻² U/kg andabout 1 U/kg. More preferably, the botulinum toxin is administered in anamount of between about 10⁻¹ U/kg and about 1 U/kg. Most preferably, thebotulinum toxin is administered in an amount of between about 0.1 unitand about 5 units. Significantly, the movement disorder alleviatingeffect of the present disclosed methods can persist for between about 2months to about 6 months when administration is of aqueous solution ofthe neurotoxin, and for up to about five years when the neurotoxin isadministered as a controlled release implant.

A further preferred method within the scope of the present invention isa method for treating a movement disorder by selecting a neurotoxin withtremor suppressant activity, choosing an intracranial target tissuewhich influences a movement disorder; and intracranially administeringto the target tissue a therapeutically effective amount of theneurotoxin selected.

Another preferred method within the scope of the present invention is amethod for improving patient function, the method comprising the step ofintracranially administering a neurotoxin to a patient, therebyimproving patient function as determined by improvement in one or moreof the factors of reduced pain, reduced time spent in bed, increasedambulation, healthier attitude and a more varied lifestyle.

The present invention encompasses a method for treating epilepsy. Themethod can comprise the step of intracranial administration of abotulinum toxin to an epileptogenic focus a patient, thereby treatingepilepsy. The botulinum toxin is a botulinum toxin types A, B, C, D, E,F or G. Preferably, the botulinum toxin is administered in an amount ofbetween about 10⁻³ U/kg and about 100 U/kg of patient weight. Thismethod can alleviate epilepsy for between about 1 month and about 5years. The botulinum toxin can be administered to a lower brain region,pontine region, mesopontine region, globus pallidus or to a thalamicregion of a brain of a patient.

The intracranial administration step can comprise implantation of acontrolled release botulinum toxin system. A detailed embodiment of thedisclosed method can comprise the step of intracranial administration ofa therapeutically effective amount of a botulinum toxin type A to anepileptogenic focus of a patient, thereby treating epilepsy.

A particular detailed method for treating epilepsy according to thepresent invention can comprise the step of intracranial administrationof a therapeutically effective amount of a botulinum toxin to aepileptogenic focus of a patient located in a thalamus of the patientbetween 3 to 6 mm posterior to the mid anterior commissure-posteriorcommissure plane, 12 mm to 16 mm lateral to the mid anteriorcommissure-posterior commissure plane, and 0 to 3 mm above the level ofthe mid anterior commissure-posterior commissure plane, thereby treatingepilepsy.

DRAWINGS

FIG. 1 illustrates preoperative placement of a frame based system forstereotactic neurosurgery for intracranial administration a botulinumtoxin to a patent with epilepsy, according to the present invention.

FIG. 2 illustrates initial intraoperative use of the system of FIG. 1.

FIG. 3 illustrates a method for positioning a patient for epilepsysurgery by temporal lobectomy induced upon stereotactic administrationof a botulinum toxin to an epileptogenic focus in the temporal lobe ofthe patient. The head of the patient is angled so that the malarprominence is the highest portion of the patient's head.

FIG. 4 illustrates a pterional incision made to expose the temporalismuscle and skull in the patient of FIG. 3.

FIG. 5 illustrates a close up of the incisional area of FIG. 4 showingretraction of the muscle and performance of a small craniotomy, so thatthe dura is opened to expose the temporal tip and a small portion of thesuprasylvian cortex.

FIG. 6 is an axial MRI image through the basal ganglia showing(highlighted) in the basal ganglia, the caudate and putamen, globuspallidus externus (“Gpe”), globus pallidus internus (“Gpi”) and thethalamus (inside the internal capsule and lateral to the thirdventricle).

FIG. 7 is a coronal image of the brain, the plane (cut) is from the topof the head to the bottom. The axons are stained black, and the neuronsare unstained. Illustrated is the putamen, globus pallidus externus,globus pallidus internus and these structures are lateral to theinternal capsule. Directly below the globus pallidus internus is theoptic tract.

DESCRIPTION

The present invention is based upon the discovery that local (i.e.intracranial) administration (as through stereotactic delivery) of abotulinum toxin (native or modified) can reduce excess electricalactivity (i.e. reduction of hyperexcitability) of an epileptic focus inthe brain, thereby treating epilepsy. As set forth herein, stereotacticmethodologies permit precise therapeutic delivery of bioactive botulinumtoxin into specific epileptic foci for the treatment of epilepsy.

A method within the scope of the present invention is primarily atreatment for intractable seizures, i.e. where surgery is indicated. Wehave surprising discovered that intracranial administration of abotulinum toxin to aberrant tissue within an identified epileptogenicfocus can be used to treat epilepsy. An intractable seizure means thatthe seizures have failed reasonable attempts at medical (drug control).Significantly, use of a botulinum toxin, unlike surgical resection, tocause a “chemical ectomy”, as described herein does not causeirreversible damage to the target neurons.

A method within the scope of the present invention can be used to treata focal epilepsy as a focal epilepsy results from a localized lesion (a“focus”) of functional abnormality. EEG can be used to localize abnormalspiking waves of a target focus followed by intracranial administrationof a botulinum toxin to non-surgically (i.e. no tissue resection orablation is carried out) downregulate the identified hyperexcitablefocus.

Botulinum toxin is too large to cross the blood brain barrier andtherefore cannot be given systemically to treat an intracranialepileptogenic brain focus. Additionally, systemic administration of abotulinum toxin can be expected to result in symptoms of botulism andpossibly death.

Without wishing to be bound by theory, a proposed physiologicalmechanism for the efficacy of a method within the scope of our inventioncan be as follows. It is hypothesized that localized delivery of abotulinum toxin (such as a botulinum toxin type A) into or in thevicinity of an active epileptic focus (or foci) disrupts thehyperexcitability of the focus thereby suppressing or limiting seizurepropagation.

The specific actions of a botulinum toxin on presynaptic nerve terminalsare well characterized at the neuromuscular junction. Briefly, botulinumtoxin binds to the presynaptic cholinergic terminals throughinteractions of its heavy chain binding domain with an, as yet,unspecified membrane receptor complex; gains entry through endocytosisthat is independent of vesicular recycling mechanisms; undergoes apH-dependent conformational shift within the endosomal vesicle thatresults in the translocation of the enzymatically active light chain tothe cytosol; blocks vesicular neurotransmitter release by cleaving theC-termini of SNAP-25 proteins involved in vesicle docking. Althoughbotulinum toxin actions in the periphery are selective for cholinergicneurons of the neuromuscular junction, experimental evidence suggeststhat the toxin is relatively non-selective in exerting actions onmammalian central nervous system neurons. Thus, botulinum toxin inhibitsneurotransmitter release from particulate preparations of brain andspinal cord (Bigalke H., et al., Tetanus toxin and botulinum A toxininhibit release and uptake of various transmitters, as studied withparticulate preparations from rat brain and spinal cord, NaunynSchmiedebergs Arch Pharmacol 1981 June; 316(3):244-51) and blockspresynaptic vesicle exocytosis in primary neuronal cultures fromhippocampus (Owe-Larsson B., et al., Distinct effects of clostridialtoxins on activity-dependent modulation of autaptic responses incultured hippocampal neurons, Eur J Neurosci 1997 August; 9(8):1773-7;Trudeau L. et al., Modulation of an early step in the secretorymachinery in hippocampal nerve terminals, Proc Natl Acad Sci USA 1998Jun. 9; 95(12):7163-8) and spinal cord (Bigalke H., et al., Botulinum Aneurotoxin inhibits non-cholinergic synaptic transmission in mousespinal cord neurons in culture, Brain Res 1985 Dec. 23;360(1-2):318-24).

While botulinum toxin has been shown to target presynaptic terminals,the toxin may be able to exert postsynaptic actions as well. Activationof the metabotropic glutamate receptor 1 (mGluR1) has been shown topotentiate N-methyl-D-aspartate (NMDA) receptor-mediated postsynapticresponses, and NMDA receptor-mediated responses have been implicated inmechanisms of synaptic plasticity and in learning and memory. It hasdemonstrated that the potentiation of NMDA responses by mGluR1activation is due to an enhanced delivery of new NMDA receptors to thepostsynaptic cell surface, through regulated vesicular exocytosis (LanJ., et al., Activation of metabotropic glutamate receptor 1 acceleratesNMDA receptor trafficking: J Neurosci 2001 Aug. 15; 21(16):6058-68). Lanet al further demonstrated that the light chain of botulinum toxin typeA attenuates the potentiating actions of mGlu1 receptor activation onNMDA receptor responses. Thus, botulinum toxin type A administration canpotentially effect both pre- and post-synaptic responses. As notedearlier, blockade or inhibition of presynaptic vesicular release atexcitatory synapses blocks neurotransmission and producesdesynchronization of network activity, such as in the epileptogenichippocampus, and leads to changes in synaptic plasticity (LTP). Thus,through inhibitory actions on synaptic vesicular release, a botulinumtoxin can produce, at least in part, a denervation of the neural networkwithin the treated focus, leading to a “functional (chemically induced)resection”, suppression of focal hyperexcitability and lasting changesin synaptic plasticity. At the same time it is significant to note that,axons coursing through the target structure without synapsing would bespared. Thus, because endocytotic processes have been characterized atpresynaptic terminals and somatodendritic cell surfaces and arenegligible or absent along axons (Huttner W., et al., Exocytotic andendocytotic membrane traffic in neurons, Curr Opin Neurobiol 1991October; 1 (3):388-92; Parton R., et al., Cell biology of neuronalendocytosis, J Neurosci Res 1993 Sep. 1; 36(1):1-9. and becausebotulinum toxin entry into neurons is mediated through endocytosis, thetoxin would not be expected to enter axons, arising from cell bodies indistant nuclei, that are strictly coursing through, but not synapsingwithin, the injected focus.

The reduction of focal hyperactivity will, expectedly, disrupt thepathological recruitment of downstream neuronal paths, resulting in asuppression of seizure propagation and yielding a desiredanticonvulsant/antiepileptic effect. This outcome is based upon ourcurrent understanding of neuroanatomical circuitry and mechanisms ofseizure propagation. In all models of cortical (and hippocampal)epileptogenesis, seizure generation and propagation is dependent uponneurotransmission (McCormick D. A., et al., On the cellular and networkbases of epileptic seizures, Annu Rev Physiol 63: 815-46; 2001). Thus,inhibition of neurotransmission within a focus would lead to inhibitionof signal transmission to target cell populations outside of the focus,and concomitant activity-dependent reduction in hyperexcitability.Additionally, there would be a reduction in ephaptic neuronalrecruitment, due to a reduction in activity-dependent field effects,resulting in decreased neuronal synchronization in perifocal tissues.Burst discharges are sensitive to inhibition resulting from thedepletion of the readily-releasable vesicle pool in presynapticterminals, as has been demonstrated in the hippocampus (Staley et al1998, Ibid). Thus, burst discharges arising from a targeted focus wouldbe subject to similar inhibition upon botulinum toxin administration,since blockade of vesicular release produces a functional effect similarto depletion of the readily-releasable vesicular pool.

Botulinum toxin injection into a epileptogenic focus can be viewed as anadjunct or alternative to resective surgery, depending upon the observedcharacteristics of the underlying hyperexcitable tissue uponlocalization and examination. As well, intrafocal toxin injection can besupplemented with standard AED pharmacotherapy postoperatively, tosuppress residual seizure activity while allowing for the toxin effectto occur.

It has been reported that “Since the thalamus and the cortex arestrongly innervated by cholinergic neurons projecting from the brainstemand basal forebrain, an imbalance between excitation and inhibition,brought about by the presence of mutant (neuronal nicotinicacetylcholine) receptors (which display an increased acetylcholinesensitivity) could generate seizures by facilitating and synchronizingspontaneous oscillations in thalmo-cortical circuits.” Raggenbass M., etal., Nicotinic receptors in circuit excitability and epilepsy, J.Neurobiol. 2002 December; 53(4):580-9. This publication clearly supportsthe proposed efficacy of the present invention.

Intracranial administration of a botulinum toxin down-regulateshyperexcitable neurons in an epileptogenic focus and can provide a curefor epilepsy due to synaptic plasticity which results in a “rewiring” ofneuronal circuitry as new neuronal circuits are established to bypassthe chemically deactivated epileptogenic focus.

Focal application of botulinum toxin can be used to treat manyindications, such as focal, generalized idiopathic, generalizedsymptomatic, and unclassified epilepsies.

Thus, the present invention is based on the discovery that significantand long lasting relief from a variety of different movement disorderscan be achieved by intracranial administration of a neurotoxin.Intracranial administration permits the blood brain barrier to bebypassed and delivers much more toxin to the brain than is possible by asystemic route of administration. Furthermore, systemic administrationof a neurotoxin, such as a botulinum toxin, is contraindicated due tothe severe complications (i.e. botulism) which can result from entry ofa botulinum toxin into the general circulation. Additionally, sincebotulinum toxin does not penetrate the blood brain barrier to anysignificant extent, systemic administration of a botulinum toxin has nopractical application to treat an intracranial target tissue.

The present invention encompasses any suitable method for intracranialadministration of a neurotoxin to a selected target tissue, includinginjection of an aqueous solution of a neurotoxin and implantation of acontrolled release system, such as a neurotoxin incorporating polymericimplant at the selected target site. Use of a controlled release implantreduces the need for repeat injections.

Intracranial implants are known. For example, brachytherapy formalignant gliomas can include stereotactically implanted, temporary,iodine-125 interstitial catheters. Scharfen. C. O., et al., HighActivity Iodine-125 Interstitial Implant For Gliomas, Int. J. RadiationOncology Biol Phys 24(4);583-591:1992. Additionally, permanent,intracranial, low dose ¹²⁵I seeded catheter implants have been used totreat brain tumors. Gaspar, et al., Permanent ¹²⁵ I Implants forRecurrent Malignant Gliomas, Int J Radiation Oncology Biol Phys 43(5);977-982:1999. See also chapter 66, pages 577-580, Bellezza D., et al.,Stereotactic Interstitial Brachytherapy, in Gildenberg P. L. et al.,Textbook of Stereotactic and Functional Neurosurgery, McGraw-Hill(1998).

Furthermore, local administration of an anti cancer drug to treatmalignant gliomas by interstitial chemotherapy using surgicallyimplanted, biodegradable implants is known. For example, intracranialadministration of 3-bis(chloro-ethyl)-1-nitrosourea (BCNU) (Carmustine)containing polyanhydride wafers, has found therapeutic application.Brem, H. et al., The Safety of Interstitial Chemotherapy withBCNU-Loaded Polymer Followed by Radiation Therapy in the Treatment ofNewly Diagnosed Malignant Gliomas: Phase I Trial, J Neuro-Oncology26:111-123:1995.

A polyanhydride polymer, GLIADEL® (Stolle R & D, Inc., Cincinnati, Ohio)a copolymer of poly-carboxyphenoxypropane and sebacic acid in a ratio of20:80 has been used to make implants, intracranially implanted to treatmalignant gliomas. Polymer and BCNU can be co-dissolved in methylenechloride and spray-dried into microspheres. The microspheres can then bepressed into discs 1.4 cm in diameter and 1.0 mm thick by compressionmolding, packaged in aluminum foil pouches under nitrogen atmosphere andsterilized by 2.2 megaRads of gamma irradiation. The polymer permitsrelease of carmustine over a 2-3 week period, although it can take morethan a year for the polymer to be largely degraded. Brem, H., et al,Placebo-Controlled Trial of Safety and Efficacy of IntraoperativeControlled Delivery by Biodegradable Polymers of Chemotherapy forRecurrent Gliomas, Lancet 345; 1008-1012:1995.

An implant can be prepared by mixing a desired amount of a stabilizedneurotoxin (such as non-reconstituted BOTOX®) into a solution of asuitable polymer dissolved in methylene chloride, at room temperature.The solution can then be transferred to a Petri dish and the methylenechloride evaporated in a vacuum desiccator. Depending upon the implantsize desired and hence the amount of incorporated neurotoxin, a suitableamount of the dried neurotoxin incorporating implant is compressed atabout 8000 p.s.i. for 5 seconds or at 3000 p.s.i. for 17 seconds in amold to form implant discs encapsulating the neurotoxin. See e.g. FungL. K. et al., Pharmacokinetics of Interstitial Delivery of Carmustine4-Hydroperoxycyclophosphamide and Paclitaxel From a BiodegradablePolymer Implant in the Monkey Brain, Cancer Research 58; 672-684:1998.

Diffusion of biological activity of a botulinum toxin within a tissueappears to be a function of dose and can be graduated. Jankovic J., etal Therapy With Botulinum Toxin, Marcel Dekker, Inc., (1994), page 150.

Local, intracranial delivery of a neurotoxin, such as a botulinum toxin,can provide a high, local therapeutic level of the toxin and cansignificantly prevent the occurrence of any systemic toxicity since manyneurotoxins, such as the botulinum toxins are too large to cross theblood brain barrier. A controlled release polymer capable of long term,local delivery of a neurotoxin to an intracranial site can circumventthe restrictions imposed by systemic toxicity and the blood brainbarrier, and permit effective dosing of an intracranial target tissue. Asuitable implant, as set forth in co-pending U.S. patent applicationSer. No. 09/587,250 entitled “Neurotoxin Implant”, allows the directintroduction of a chemotherapeutic agent to a brain target tissue via acontrolled release polymer. The implant polymers used are preferablyhydrophobic so as to protect the polymer incorporated neurotoxin fromwater induced decomposition until the toxin is released into the targettissue environment.

Local intracranial administration of a botulinum toxin, according to thepresent invention, by injection or implant to e.g. the cholinergicthalamus presents as a superior alternative to thalamotomy in themanagement of inter alia tremor associated with Parkinson's disease.

A method within the scope of the present invention includes stereotacticplacement of a neurotoxin containing implant using theRiechert-Mundinger unit and the ZD (Zamorano-Dujovny) multipurposelocalizing unit. A contrast-enhanced computerized tomography (CT) scan,injecting 120 ml of omnipaque, 350 mg iodine/ml, with 2 mm slicethickness can allow three dimensional multiplanar treatment planning(STP, Fischer, Freiburg, Germany). This equipment permits planning onthe basis of magnetic resonance imaging studies, merging the CT and MRItarget information for clear target confirmation.

The Leksell stereotactic system (Downs Surgical, Inc., Decatur, Ga.)modified for use with a GE CT scanner (General Electric Company,Milwaukee, Wis.) as well as the Brown-Roberts-Wells (BRW) stereotacticsystem (Radionics, Burlington, Mass.) can be used for this purpose.Thus, on the morning of the implant, the annular base ring of the BRWstereotactic frame can be attached to the patient's skull. Serial CTsections can be obtained at 3 mm intervals though the (target tissue)region with a graphite rod localizer frame clamped to the base plate. Acomputerized treatment planning program can be run on a VAX 11/780computer (Digital Equipment Corporation, Maynard, Mass.) using CTcoordinates of the graphite rod images to map between CT space and BRWspace.

Within wishing to be bound by theory, a further mechanism can beproposed for the therapeutic effects of a method practiced according tothe present invention. Thus, a neurotoxin, such as a botulinum toxin,can inhibit neuronal exocytosis of several different CNSneurotransmitters, in particular acetylcholine. It is known thatcholinergic neurons are present in the thalamus. Additionally,cholinergic nuclei exist in the basal ganglia or in the basal forebrain,with protections to motor and sensory cerebral regions. Thus, targettissues for a method within the scope of the present invention caninclude neurotoxin induced, reversible denervation of intracranial motorareas (such as the thalamus) as well as brain cholinergic systemsthemselves (such as basal nuclei) which project to the intracranialmotor areas. For example, injection or implantation of a neurotoxin to acholinergically innervated thalamic nuclei (such as Vim) can result in(1) downregulation of Vim activity due to the action of the toxin uponcholinergic terminals projecting into the thalamus from basal ganglia,and; (2) attenuation of thalamic output due to the action of the toxinupon thalamic somata, both cholinergic and non-cholinergic, therebyproducing a chemical thalamotomy.

Preferably, a neurotoxin used to practice a method within the scope ofthe present invention is a botulinum toxin, such as one of the serotypeA, B, C, D, E, F or G botulinum toxins. Preferably, the botulinum toxinused is botulinum toxin type A, because of its high potency in humans,ready availability, and known use for the treatment of skeletal andsmooth muscle disorders when locally administered by intramuscularinjection. Botulinum toxin type B is a less preferred neurotoxin to usein the practice of the disclosed methods because type B is known to havea significantly lower potency and efficacy as compared, to type A, isnot readily available, and has a limited history of clinical use inhumans. Furthermore, the higher protein load with regard to type B cancause immunogenic reaction to occur with development of antibodies tothe type B neurotoxin.

The amount of a neurotoxin selected for intracranial administration to atarget tissue according to the present disclosed invention can be variedbased upon criteria such as the movement disorder being treated, itsseverity, the extent of brain tissue involvement or to be treated,solubility characteristics of the neurotoxin toxin chosen as well as theage, sex, weight and health of the patient. For example, the extent ofthe area of brain tissue influenced is believed to be proportional tothe volume of neurotoxin injected, while the quantity of the tremorsuppressant effect is, for most dose ranges, believed to be proportionalto the concentration of neurotoxin injected. Methods for determining theappropriate route of administration and dosage are generally determinedon a case by case basis by the attending physician. Such determinationsare routine to one of ordinary skill in the art (see for example,Harrison's Principles of Internal Medicine (1998), edited by AnthonyFauci et al., 14^(th) edition, published by McGraw Hill).

We have found that a neurotoxin, such as a botulinum toxin, can beintracranially administered according to the present disclosed methodsin amounts of between about 10⁻³ U/kg to about 10 U/kg. A dose of about10⁻³ U/kg can result in an epileptic tremor suppressant effect ifdelivered to a small intracranial nuclei. Intracranial administration ofless than about 10⁻³ U/kg does not result in a significant or lastingtherapeutic result. An intracranial dose of more than 10 U/kg of aneurotoxin, such as a botulinum toxin, poses a significant risk ofdenervation of sensory or desirable motor functions of neurons adjacentto the target.

A preferred range for intracranial administration of a botulinum toxin,such as botulinum toxin type A, so as to achieve a tremor suppressanteffect in the patient treated is from about 10⁻² U/kg to about 1 U/kg.Less than about 10⁻² U/kg can result in a relatively minor, though stillobservable, tremor suppressant effect. A more preferred range forintracranial administration of a botulinum toxin, such as botulinumtoxin type A, so as to achieve an antinociceptive effect in the patienttreated is from about 10⁻¹ U/kg to about 1 U/kg. Less than about 10⁻¹U/kg can result in the desired therapeutic effect being of less than theoptimal or longest possible duration. A most preferred range forintracranial administration of a botulinum toxin, such as botulinumtoxin type A, so as to achieve a desired tremor suppressant effect inthe patient treated is from about 0.1 units to about 100 units.Intracranial administration of a botulinum toxin, such as botulinumtoxin type A, in this preferred range can provide dramatic therapeuticsuccess.

The present invention includes within its scope the use of anyneurotoxin which has a long duration tremor suppressant effect whenlocally applied intracranially to the patient. For example, neurotoxinsmade by any of the species of the toxin producing Clostridium bacteria,such as Clostridium botulinum, Clostridium butyricum, and Clostridiumberatti can be used or adapted for use in the methods of the presentinvention. Additionally, all of the botulinum serotypes A, B, C₁, D, E,F and G can be advantageously used in the practice of the presentinvention, although type A is the most preferred and type B the leastpreferred serotype, as explained above. Practice of the presentinvention can provide a tremor suppressant effect, per injection, for 3months or longer in humans.

Significantly, a method within the scope of the present invention canprovide improved patient function. “Improved patient function” can bedefined as an improvement measured by factors such as a reduced pain,reduced time spent in bed, increased ambulation, healthier attitude,more varied lifestyle and/or healing permitted by normal muscle tone.Improved patient function is synonymous with an improved quality of life(QOL). QOL can be assesses using, for example, the known SF-12 or SF-36health survey scoring procedures. SF-36 assesses a patient's physicaland mental health in the eight domains of physical functioning, rolelimitations due to physical problems, social functioning, bodily pain,general mental health, role limitations due to emotional problems,vitality, and general health perceptions. Scores obtained can becompared to published values available for various general and patientpopulations.

As set forth above, we have discovered that a surprisingly effective andlong lasting treatment of a focal epilepsy can be achieved byintracranial administration of a neurotoxin to an afflicted patient. Inits most preferred embodiment, the present invention is practiced byintracranial injection or implantation of botulinum toxin type A.

The present invention does include within its scope: (a) neurotoxinobtained or processed by bacterial culturing, toxin extraction,concentration, preservation, freeze drying and/or reconstitution and;(b) modified or recombinant neurotoxin, that is neurotoxin that has hadone or more amino acids or amino acid sequences deliberately deleted,modified or replaced by known chemical/biochemical amino acidmodification procedures or by use of known host cell/recombinant vectorrecombinant technologies, as well as derivatives or fragments ofneurotoxins so made.

Botulinum toxins for use according to the present invention can bestored in lyophilized, vacuum dried form in containers under vacuumpressure or as stable liquids. Prior to lyophilization the botulinumtoxin can be combined with pharmaceutically acceptable excipients,stabilizers and/or carriers, such as albumin. The lyophilized materialcan be reconstituted with saline or water.

EXAMPLES

The following examples set forth specific methods encompassed by thepresent invention to treat a movement disorder and are not intended tolimit the scope of the invention.

Example 1 Intracranial Target Tissue Localization and Methodology

Stereotactic procedures can be used for precise intracranialadministration of neurotoxin in aqueous form or as an implant to desiredtarget tissue. Thus, intracranial administration of a neurotoxin totreat a drug resistant tremor (i.e. a resting tremor, such as can occurin Parkinson's disease, or an action tremor, such as essential tremor),multiple sclerosis tremors, post traumatic tremors, post hemiplegictremors (post stroke spasticity), tremors associated with neuropathy,writing tremors and epilepsy can be carried out as follows.

A preliminary MRI scan of the patient can be carried out to obtain thelength of the anterior commissure-posterior commissure line and itsorientation to external bony landmarks. The base of the frame can thenbe aligned to the plane of the anterior commissure-posterior commissureline. CT guidance is used and can be supplemented with ventriculography.The posterior commissure can be visualized on 2-mm CT slices and used asa reference point. Where the target injection site is the basal part ofthe ventral intermedius nucleus of the ventrolateral thalamus, averagecoordinates are 6.5 mm anterior to the posterior commissure, 11 mmlateral to the third ventricular wall and 2 mm above the anteriorcommissure-posterior commissure line. This location is not expected toencroach on the sensory thalamus or on a subthalamic region.

Physiological corroboration of target tissue localization can be by useof high and low frequency stimulation through an electrode accompanyingor incorporated into the long needle syringe used. A thermistorelectrode 1.6 mm in diameter with a 2 mm exposed tip can be used(Radionics, Burlington, Mass.). With electrode high frequencystimulation (75 Hz) paraesthetic responses can be elicited in theforearm and hand at 0.5-1.0 V using a Radionics lesion generator(Radionics Radiofrequency Lesion Generator Model RFG3AV). At lowfrequency (5 Hz) activation or disruption of tremor in the affected limboccurred at 2-3 V. With the methods of the present invention, theelectrode is not used to create a lesion. Following confirmation oftarget tissue localization, a neurotoxin can be injected, therebycausing a reversible, chemical thalamotomy. A typical injection is thedesired number of units (i.e. about 0.1 to about 5 units of a botulinumtoxin type A complex in about 0.1 ml to about 0.5 ml of water or saline.A low injection volume can be uses to minimize toxin diffusion away fromtarget. Typically, the neurotransmitter release inhibition effect can beexpected to wear off within about 2-4 months. Thus, an alternateneurotoxin format, neurotoxin incorporated within a polymeric implant,can be used to provide controlled, continuous release of therapeuticamount of the toxin at the desired location over a prolonged period.(i.e. from about 1 year to about 6 years), thereby obviating the needfor repeated toxin injections.

Several methods can be used for stereotactically guided injection of aneurotoxin to various intracranial targets, such as the subthalamicnucleus (STN) for treatment of Parkinson's disease (Parkinson'sdisease). Thus a stereotactic magnetic resonance (MRI) method relying onthree-dimensional (3D) T1-weighted images for surgical planning andmultiplanar T2-weighted images for direct visualization of the STN,coupled with electrophysiological recording and injection guidance forunilateral or bilateral STN injection can be used. See e.g. Bejjani, B.P., et al., Bilateral Subthalamic Stimulation for Parkinson's Disease byUsing Three-Dimensional Stereotactic Magnetic Resonance Imaging andElectrophysiological Guidance, J Neurosurg 92(4); 615-25:2000. The STNscan be visualized as 3D ovoid biconvex hypointense structures located inthe upper mesencephalon. The coordinates of the centers of the STNs canbe determined with reference to the patient's anteriorcommissure-posterior commissure line by using as a landmark, theanterior border of the red nucleus.

Electrophysiological monitoring through several parallel tracks can beperformed simultaneously to define the functional target accurately.Microelectrode recording can identify high-frequency, spontaneous,movement-related activity and tremor-related cells within the STNs.Neurotoxin injection into the STN can improve contralateral rigidity andakinesia and suppress tremor when present. The central track, which isdirected at the predetermined target by using MRI imaging, can beselected for neurotoxin injection. No surgical complications areexpected. The patient can show significantly improved parkinsonian motordisability in the “off” and “on” medication states and use ofantiparkinsonian drug treatment can be dramatically reduced as is theseverity of levodopa-induced dyskinesias and motor fluctuations.

Computer-aided atlas-based functional neurosurgery methodology can beused to accurately and precisely inject the desired neurotoxin orimplant a neurotoxin controlled release implant. Such methodologiespermit three-dimensional display and real-time manipulation of cerebralstructures. Neurosurgical planning with mutually preregistered multiplebrain atlases in all three orthogonal orientations is therefore possibleand permits increased accuracy of target definition for neurotoxininjection or implantation, reduced time of the surgical procedure bydecreasing the number of tracts, and facilitates planning of moresophisticated trajectories. See e.g. Nowinski W. L. et al.,Computer-Aided Stereotactic Functional Neurosurgery Enhanced by the Useof the Multiple Brain Atlas Database, IEEE Trans Med Imaging 19(1);62-69:2000.

Example 2 Treatment of Parkinson's Disease with Botulinum Toxin Type A

A 64 year old right-handed male presents with pronounced tremor of theextremities, bradykinesia, rigidity and postural changes such that hefrequently falls. A prominent pill rolling tremor is noted in his righthand. Stroke is ruled out and it is noted that the symptoms are worse onhis right side. Diagnosis of Parkinson's disease is made. Using CAT scanor MRI assisted stereotaxis, as set forth in Example 1 above, 2 units ofa botulinum toxin type A (such as BOTOX® or about 8 units of DYSPORT®)is injected into the left side of the globus pallidus. The patient isdischarged within 48 hours and with a few (1-7) days enjoys significantimprovement of the parkinsonian motor symptoms more clearly on theright, but also on his left side. His dyskinesias almost completelydisappear. The motor disorder symptoms of Parkinson's disease remainsignificantly alleviated for between about 2 to about 6 months. Forextended therapeutic relief, one or more polymeric implantsincorporating a suitable quantity of a botulinum toxin type A can beplaced at the target tissue site.

Example 3 Treatment of Parkinson's Disease with Botulinum Toxin Type B

A 68 year left handed male presents with pronounced tremor of theextremities. bradykinesia, rigidity and postural changes such that hefrequently falls. A prominent pill rolling tremor is noted on his leftside. Stroke is ruled out and it is noted that the symptoms are worse onhis left side. Diagnosis of Parkinson's disease is made. Using CAT scanor MRI assisted stereotaxis, as set forth in Example 1 above, from 10 toabout 50 units of a botulinum toxin type B preparation (such asNEUROBLOC® or INERVATE™) is injected into the right side of the globuspallidus. The patient is discharged within 48 hours and with a few (1-7)days enjoys significant improvement of the parkinsonian motor symptomsmore clearly on the left, but also on his right side. His dyskinesiasalmost completely disappear. The motor disorder symptoms of Parkinson'sdisease remain significantly alleviated for between about 2 to about 6months. For extended therapeutic relief, one or more polymeric implantsincorporating a suitable quantity of a botulinum toxin type B can beplaced at the target tissue site.

Example 4 Treatment of Parkinson's Disease with Botulinum Toxin TypesC₁-G

A female aged 71 is admitted with uncontrollable and frequent tremor.From 0.1 to 100 units of a botulinum toxin type C₁, D, E, F or G isinjected unilaterally into the ventrolateral thalamus for the disablingtremors. CAT scan or MRI assisted stereotaxis, as set forth in Example 1above, supplemented by ventriculography is used. The patient isdischarged within 48 hours and with a few (1-7) days enjoys significantremission of tremors which remain significantly alleviated for betweenabout 2 to about 6 months. For extended therapeutic relief, one or morepolymeric implants incorporating a suitable quantity of a botulinumtoxin type C₁, D, E, F or G can be placed at the target tissue site.

Example 5 Treatment of Dystonia with Botulinum Toxin Type A

A 16 year old male child with severe, incapacitating dystonia, secondaryto cranial trauma, affecting the proximal limb muscles is a candidatefor unilateral thalamotomy on the left side, bilateral thalamotomycarrying a high risk of iatrogenic dysarthria and pseudubulbar effects.The patient has failed to respond or has become unresponsive totranscutaneous nerve stimulation, feedback display of the EMG andanticholinergics. The dystonia is relatively stable, the patient issufficiently fit to withstand surgery and is significantly disabled withdistal phasic and tonic limb dystonia.

A suitable stereotactic frame can be applied to the head with localanesthetic and ventriculography and stereotactic MRI can be performed.The stereotactic coordinates of the anterior commissure (AC) and theposterior commissure (PC) can be determined by using the computersoftware in the scanner. PC based software can be used to redraw thesagittal brain maps from the Schaltenbrand and Bailey and Schaltenbrandand Wahren atlases, stretched or shrunk as needed to the AC-PC distanceof the patient and ruled in stereotactic coordinates for the actualapplication of the frame to the patient's head. The target sites areselected, their coordinates are read off and appropriate frame settingsare made. A burr hole or twist-drill hole can be made at or rostral tothe coronal suture in the same sagittal plane as the target. This canfacilitate plotting the physiological data used for target corroborationsince the electrode trajectories traverse a single sagittal plane. Theventrocaudal nucleus of the thalamus (Vc) can be selected as aphysiological landmark, lying 15 mm from the midline. The Vc can beeasily recognized by recording individual tactile cells within it withtheir discrete receptive fields or by inducing paresthesias withstimulation in discreet projected fields.

A microelectrode recording needle (such a used for single fiberelectromyographic recording having an approximately 25 micron diameterrecording electrode) can be located within the bore of a microsyringeand is advanced toward the expected tactile representation of thefingers in the Vc and continuous recording is carried out to search foridentifiable neurons. Microstimulation can be performed everymillimeter, beginning about 10 mm above and extending to a variabledistance below the target. If the first microelectrode trajectoryenters, for example, the tactile representation of the lips of a patientwith upper limb dystonia, a second trajectory can be carried out 2 mmmore lateral. Upon encountering lower limb responses, the nexttrajectory can be made 2 mm more medial. Once the tactile representationof the hand is found, the next trajectory can be made 2 mm rostral toit, where recording reveals kinesthetic neurons that respond to bendingof specific contralateral joints or pressure on specific contralateralsites. If dystonia is confined to the leg, the process described abovecan be aimed at the thalamic representation for the leg.

Upon microstimulation localization of the stereotactically-MRI guidedrecording/stimulating needle electrode to the target, a neurotoxinimplant can be injected. The implant can comprise a neurotoxin, such asa of botulinum toxin type A, incorporated within biodegradable polymericmicrospheres or a biodegradable pellet, either implant format containingabout 20 total units (about 1 ng) of the toxin with implantcharacteristics of continuous release over a period of at least aboutfour years of a therapeutic level of the toxin at point of the implantrelease site and for a radius of about 2-3 mm on each side of the targetsite. The implant can release about 1 unit of toxin essentiallyimmediately and further amounts of about one unit cumulatively oversubsequent 2-4 months periods.

The patient's dystonic contractions can subside almost immediately, andcan remain substantially alleviated for between about 2 months to about6 months per toxin injection or for between about 1 to 5 years dependingupon the particular release characteristics of the implant polymer andthe quantity of neurotoxin loaded therein.

Example 6 Treatment of Dystonia with Botulinum Toxin Types B-G

The patient of example 5 above can be equivalently treated using thesame protocol and approach to target with between about 1 unit and about1000 units of a botulinum toxin type B, C₁, D, E, F or G in aqueoussolution or in the form of a suitable neurotoxin implant. With such atreatment, the dystonic contractions subside within 1-7 days, and remainsubstantially alleviated for between about 2-6 months per toxininjection or for between about 1 to 5 years depending upon theparticular release characteristics of the implant polymer and thequantity of neurotoxin loaded therein.

Example 7 Treatment of Tremor With Botulinum Toxin Type A

A 44 year old male presents with severe incapacitating tremor of threeyears duration which disrupts his activities of daily living. There isalso asymmetry of the motor symptoms between the two side of the bodyand levodopa has induced dyskinesia in the extremities. Tremor cells areidentified by stereotactic examination of the effect upon the tremor byelectrical stimulation of the proposed target cell. The effect ofstimulation is noted to inhibit the tremor. Stereotactic guided (as inExample 1) implant placement can be made at a site about 14 to 15 mmfrom the midline and 2-3 mm above the AC-PC line in the middle ofkinesthetic and/or voluntary tremor cells. The target site can be the VLor Vi.

The implant can be either an aqueous solution of botulinum toxin type Aincorporated within biodegradable polymeric microspheres or botulinumtoxin type A biodegradable pellet, either implant format containingabout 20 total units (about 1 ng) of the toxin with implantcharacteristics of continuous release over a period of at least aboutfour years of a therapeutic level of the toxin at point of the implantrelease site and in about 2-3 mm on each side. The implant can releaseabout 1 unit of toxin essentially immediately and further amounts ofabout one unit cumulatively over subsequent 2-4 months periods.

The patient's tremors can subside within 1-7 days, and can remainsubstantially alleviated for between about 2 months to about 6 monthsper toxin injection or for between about 1 to 5 years depending upon theparticular release characteristics of the implant polymer and thequantity of neurotoxin loaded therein. Notably, there can be significantattenuation of distal limb movements, both phasic and tonic on the rightside.

Example 8 Treatment of Tremor with Botulinum Toxin Types B-G

The patient of example 7 above can be equivalently treated using thesame protocol and approach to target with between about 1 unit and about1000 units of a botulinum toxin type B, C₁, D, E, F or G in aqueoussolution or in the form of a suitable neurotoxin implant. With such atreatment, the tremors can subside within 1-7 days, and can remainsubstantially alleviated for between about 2-6 months per toxininjection or for between about 1 to 5 years depending upon theparticular release characteristics of the implant polymer and thequantity of neurotoxin loaded therein.

Example 9 Treatment of Epilepsy with Botulinum Toxin Type A

A right handed, female patient age 22 presents with a history ofepilepsy. Based upon MRI and a study of EEG recording, a diagnosis oftemporal lobe epilepsy is made. An implant which provides about 5-50units of a neurotoxin (such as a botulinum toxin type A) can be insertedat (or alternately 1-10 units of a botulinum toxin type A aqueoussolution [less than 0.1 milliliter volume] can be infused or injectedinto) an identified epileptogenic focus located in the anterior part ofthe temporal lobe, 5-6 cm from the tip of the lobe along the middletemporal gyrus with a unilateral approach to the nondominant, lefthemisphere. The epileptic seizures can be substantially reduced almostimmediately, and remain substantially alleviated thereafter.

Administration of the botulinum toxin is carried out so as to reduce thelikelihood of dosing non-epileptized cerebral tissues. Thus, toxinadministration is not to tissues outside the target focus in thetemporal lobe where the ictal discharges responsible for the seizuresare localized. This can be carried out by respecting certain anatomiclandmarks to thereby avoid visual and language disorders so that dosingspares temporal language cortical region and optic radiations. Hence,the posterior limits for dosing (i.e. for the extent of the chemicallobectomy caused by the botulinum toxin) is 5-6 cm from the tip of thelobe along the middle temporal gyrus when operating on the nondominanthemisphere, and 4-4.5 cm on the dominant side. Additionally, only thefirst 2 cm of the superior gyrus are within the toxin dose field.

Example 10 Treatment of Epilepsy with Botulinum Toxin Types B-G

The patient of example 9 above or in examples 11-20 can be equivalentlytreated using the same protocol and approach to target with betweenabout 1 unit and about 1000 units of a botulinum toxin type B, C₁, D, E,F or G in aqueous solution or in the form of a suitable neurotoxinimplant. With such a treatment, the epileptic seizures subside within afew minutes or hours and can remain substantially alleviated thereafter.

It is concluded that neurotoxin injection or implantation of acontrolled release neurotoxin implant according to the methods of thepresent invention, with the aid of 3D MR imaging andelectrophysiological guidance, can be a safe and effective therapy forpatients suffering from various movement disorders, such as severe,advanced non-AED responsive epilepsy. Suitable patients include thosewho have become largely if not entirely refractory to chemotherapy priorto intracranial neurotoxin administration as set forth herein.

Example 11 Treatment of Epilepsy by Administration of a Botulinum Toxinto an Epileptogenic Focus Located in the Thalamus

A 34 year old male patient present with intractable epilepsy andfrequent limb tremors. To identify an abnormal area of cortex from whichthe seizures originate a noninvasive, presurgical evaluation isconducted with the patient's consent, comprising the taking of adetailed clinical history, followed by a physical examination,neuro-imaging (magnetic resonance imaging (MRI), functional imaging tovisualize alterations in cerebral metabolism using Positron EmissionTomography (PET), and Single Photon Emission Computerized Tomography(SPECT)), 24 hour intensive video-EEG monitoring, neuropsychologicaltesting and assessment of psychosocial functioning.

The evaluation locates a focal origin of the seizures, as a primaryepileptogenic region. Standard preoperative blood tests are performedwith special attention to platelets, bleeding time, PT and PTT. Thepatients is advised to discontinue aspirin and other non-steroidalanti-inflammatory agents at least 5 days prior to surgery. The patientbegins a fast the evening before surgery.

The toxin induced thalamotomy is performed under local anesthesia andrequires the full cooperation of the patient therefore theintraoperative use of sedating agents is avoided. Intravenous access isestablished ipsilateral to the planned botulinum toxin inducedthalamotomy to allow complete freedom of movement in the extremity ofinterest and oxygen is supplied by nasal cannula. EKG, pulse oximetryand BP is monitored but an arterial line is not inserted. Blood pressureis maintained in the normal range for the patient. Bladdercatheterization is not performed.

An MRI compatible stereotactic frame is affixed to the cranial vaultafter infiltration of the pin insertion sites with 1% lidocaine with1/200,000 epinephrine. The pin insertion sites are chosen to avoidartifact through the planned axial imaging sections of interest. Theframe (illustrated by FIG. 1) is placed as symmetrically as possible onthe head to minimize rotation and lateral tilt. This ensures that anychanges made in electrode position intraoperatively are entirely in theplanned direction. The instrument used is the CRW frame (Radionics,Burlington, Mass.). Following frame placement (as illustrated by FIG.2), the patient is taken to the MRI scanner where sagittal T1-weightedimages are obtained first. These images are used to identify theanterior commissure (AC) and the posterior commissure (PC) and tomeasure the AC-PC length. Next T1-weighted (TR 400, TE 12/Fr, FOV 30×30,2 NEX, 3 mm thickness) axial images are obtained through the basalganglia so that these images are parallel to the AC-PC plane. Additionalimages in the coronal plane with fast spin echo inversion (FSE IR)recovery sequences to accentuate the gray-white matter borders of thethalamus and internal capsule are also used. The spatial accuracy of thestereotactic frame in the scanner is verified with phantoms.

Stereotactic CT imaging is also carried out to enhance targetingaccuracy. Thus, after MR imaging, the patient is taken to the CT scannerand after the appropriate localizer is placed, and axial CT images(DFOV, 1.0-1.5 mm thickness) roughly parallel to the intercommissuralline and through the area of interest are obtained. The AC/PC line isidentified and used for initial target identification.

The target focus is located at a point 4 to 5 mm posterior to the midanterior commissure-posterior commissure (AC-PC) plane, 13.5 to 15 mmlateral to the midline, and 0 to 1 mm above the level of theintercommisural plane. When the third ventricle is dilated, we add 1-2mm to the lateral dimension.

Once the target point has been calculated, the patient is brought to theoperating room. A single dose of an appropriate prophylactic antibioticis given. The patient is placed in a comfortable position (FIG. 3) andthe frame is fixed to the operating table with the head only slightlyelevated above the chest to avoid air embolism. A small patch of hair isshaved over the appropriate frontal region and the area is then preparedand draped. After infiltration of the scalp with 1% lidocaine with1/200,000 epinephrine, a 2.5 cm parasagittal incision (FIG. 4) is madeand a burr hole placed 2.0 cm from the midline at the level of thecoronal suture. The dura is coagulated and opened and a small pialincision made, so that the dura is opened to expose the temporal tip anda small portion of the suprasylvian cortex (FIG. 5). Cortical veins areavoided to permit atraumatic introduction of the electrode. At thispoint the stereotactic arc is brought into the target position and theelectrode guide tube is lowered into the burr hole directly over thepial incision. The skin is then temporarily closed around the guide tubewith nylon sutures to prevent excessive loss of CSF and brain settling.

Since there are individual differences in thalamic anatomy, initialtarget selection is only approximate and final targeting is performedusing micro-electrode recordings, and macrostimulation. The initial passis performed with a micro-electrode to obtain spontaneousneurophysiologic data and then this is followed by themacrostimulation/lesioning electrode. True microelectrodes are used(made of tungsten), which allow for the discrimination of singleneurons. The electrodes are connected via short leads to a preamplifier.The signal from the preamplifier is then filtered, amplified, and passedthrough a window discriminator.

Microelectrode recordings in the ventrolateral thalamus reflect theconnectivity of the various nuclei. Recordings in the Vop nucleusreveals voluntary cells that are less noisy than those in Vim(ventrointermedius nucleus of the thalamus) or VC (ventrocaudalis).These cells change their firing rate in advance or at the beginning oftheir related movements. Other cells may increase their firing shortlybefore the movement while others may show a decreased rate or becomerhythmic at the onset or completion of a movement. Recordings in Vimreveal moderately noisy high voltage neurons which respond tocontralateral passive joint movement, squeezing of muscle bellies, orpressure on deep structures such as tendons. This patient has tremorsand kinesthetic cells fire rhythmically at the tremor frequency.Microelectrode recordings in VC reveal very noisy spontaneous activityand many high voltage cells. These cells respond to superficial lighttough such as light brushing of the skin or a puff of air. The cellsrespond faithfully without fatigue. The largest volume of VC is occupiedby tactile cells representing the face and manual digits. The floor ofthe thalamus is difficult to discern but is identified by the suddenloss of spontaneous neuronal activity as the microelectrode leaves thegray matter of the thalamus and enters the white matter of the zonaincerta. Careful analysis of the neuronal activity of these various celltypes confirms that the appropriate target in Vim thalamus is selected.

Macrostimulation is also used to delineate the optimal location fortoxin administration. A commercially available lesion generator(Radionics, Burlington, Mass.), is used for impedance monitoring,stimulation, but is not used for lesioning. Based on the stereotacticcoordinates, a side-seeking (SSE) macroelectrode (Radionics, Burlington,Mass.) with a 4 mm×1.8 mm uninsulated tip is introduced under impedancemonitoring. The impedance drops by about 100 when the gray matter of thebasal ganglia/thalamus is reached. Within the electrode tip is a muchsmaller electrode (2 mm×0.5 mm) that can be extruded in small incrementsso that without moving the parent electrode shaft, exploration medially,laterally and posteriorly at any angle is possible. It is through thissmaller electrode that stimulation is performed. Stimulation isperformed with square wave pulses at 0.5 to 2.0 volts with a frequencyof 2 Hz to obtain motor thresholds and at 50 to 75 Hz to assess foramelioration of symptoms or sensory responses. A botulinum toxin type Acan be injected into the target tissue through a stereotactically placed30 gauge stainless steel tube using the method set forth by Levy R., etal., Lidocaine and muscimol microinjections in subthalamic nucleusreverse parkinsonian symptoms, Brain (2001), 124, 2105-2118. Thus, theinjection cannula can be connected to a 10-15 cm piece of polyethylenetubing with a inside diameter of 0.58 mm and sealed with epoxy glue. Theaqueous toxin can be preloaded in the cannula and polyethylene tubing.

The toxin induced thalamotomy target is the Vim nucleus andoccasionally, the mere introduction of the electrode can reduce anepileptic tremor indicating that the electrode is in good position. Moreoften, due to individual variation and the small size of Vim, theelectrode can be in a suboptimal position and require adjustment. Ourgoal is to place toxin within Vim, directly anterior to the appropriatesomatotopic area in VC and medial to the internal capsule withoutencroaching on either structure. Fortunately, intraoperative stimulationand microelectrode recording allows for differentiation of the internalcapsule, Vop (ventraloralis posterior nucleus), Vim and VC nuclei basedon their physiologic responses. If the electrode is placed tooanteriorly in the Vop nucleus, low frequency stimulation can inducemovement in the contralateral limbs. This movement is focal atthreshold, beginning at one joint and involving greater parts of thecontralateral limbs as stimulation intensity is increased.

Since Vim is thought to be the relay nucleus for kinesthetic sensationand VC the relay nucleus for superficial tactile sensation, highfrequency stimulation can generally reflect this difference. Stimulationof the Vim usually elicits contralateral parasthesias at higherthresholds than those obtained in the VC nucleus. Vim stimulation canalso induce a proprioceptive sensation that a contralateral limb ismoving without any actual movement having taken place and may alsoinduce peculiar sensations of vertigo, fainting, or dread but this isgenerally seen when the electrode is too inferior. It is important todistinguish Vim from VC for optimal catheter positioning. High frequencystimulation of the VC nucleus always causes contralateral parasthesias.However, the threshold (0.25-0.5 volts) for inducing parasthesias isusually much lower than that of the Vim nucleus. Consequently, lowthreshold parasthesias of the fingertips or mouth indicate that theelectrode is too posterior and needs to be moved anteriorly. Sustainedsuprathreshold stimulation within VC may cause parasthesias that areunbearably intense. There is a clear medial-to-lateral somatotopy withinVC with neurons representing the face most medial, those representingthe lower limbs more lateral and those representing the upper extremityand hand intermediate. The definition of this somatotopic distributionis important as the toxin induced lesion in Vim should be made directlyanterior to the appropriate site in the VC nucleus.

Another indication that the electrode is in good position relates totremor response. Low frequency (2 Hz) stimulation within Vim usuallycauses driving of the tremor whereas high frequency (50 Hz) stimulationcauses amelioration of the tremor. Suppression of epileptic tremor with0.5-2.0 volts is the goal and indicates accurate targeting. In additionto the anterior-posterior differences between the nuclei, there is alsoa medial-to-lateral somatotopy within Vim. The face and mouth arerepresented most medially while the lower extremities are representedmore laterally near the internal capsule. A botulinum toxin inducedlesion is directed at the site corresponding to the most severe tremor.Lesions for tremor involving the upper extremity are placed slightlymore medial than lesions for tremor involving the lower extremities. Useof the side exploring electrode allows for simplified exploration ofthis somatotopic organization because the electrode can be partiallywithdrawn, rotated, and reinserted thereby eliminating the need forcomplete repositioning.

It is determined that the electrode is correctly positioned at thephysiologic target site, as the mere presence of the electrode in Vim,and/or high frequency stimulation causes a reduction in the epileptictremor. In addition, stimulation is also used to ensure that there is noevidence or neurologic impairment with particular attention being paidto speech and motor difficulties. The FIG. 6 MRI image shows the generallocation of the thalamus (further delineated by FIG. 7). The optic tractis avoided to prevent vision deficits.

Once the target has been confirmed, a test infusion of 1 μl of 0.1 Unita botulinum toxin type A in non-preserved saline is made. During thistime, the patient is tested neurologically for contralateral motordexterity and sensation along with verbal skills. As there no seizureinduced and no neurologic problems noted, a further infusion of 2 μl of0.2 Units of the toxin is carried out. During the toxin infusion theneurological status of the patient is continuously monitored andinfusion is halted if any impairment or change is noted. If completeabolition of the tremor has not been accomplished, then the infusionvolume is increased as guided by the intraoperative physiologicresponses and recordings.

After toxin infusion, the electrode and it's accompanying catheter iswithdrawn, and the incision is irrigated. The burr hole is filled withGelfoam and bone dust and the scalp is closed using a layer of inverted3.0 Vicryl sutures for the galea and 4.0 nylon for the skin. The frameis removed and a dry sterile dressing is placed.

Bilateral toxin induced thalamotomies are generally associated with ahigh complication rate and not undertaken.

After a brief period of observation the patient is returned directly tohis room. The patient continues with his preoperative medications andoutside of mild analgesics, no other medications are given. Given thathemorrhage is an important cause of serious morbidity, good control ofblood pressure in the perioperative period is maintained. An MRI scan isobtained within the first 24 hours to assess toxin location and toexclude perioperative complications. If neurologically stable, thepatient is discharged on the first postoperative day. Sutures areremoved one week after surgery. A short course of outpatientrehabilitation therapy is provided to optimize functional recovery ofthe affected limb. The intracranial botulinum toxin therapy cures thepatient as he remains free of any epileptic seizures for between sixmonths and five years.

Example 12 Treatment of Epilepsy by Administration of a Botulinum Toxinto an Epileptogenic Focus Located in the Globus Pallidus

Patient DE, a 49 year old male is evaluated for pallidotomy through anextensive preoperative assessment. Before surgery, the patient is keptwithout medications for 12 hours. This is done to ensure that patient isin his OFF state in order to minimize involuntary movements duringimaging and to more readily assess the effects of surgery. Prior toplacement of the frame the patient is mildly sedated with a short actingsedative, such as midazolam or propofol.

Pallidotomy is performed under local anesthesia and requires the fullcooperation of the patient therefore the intraoperative use of sedatingagents is avoided. Intravenous access is established ipsilateral to theplanned pallidotomy to allow complete freedom of movement in theextremity of interest and oxygen is supplied by nasal cannula. EKG,pulse oximetry and BP is monitored but an arterial line is not inserted.Blood pressure is maintained in the normal range for the patient.Bladder catheterization is not performed. An MRI compatible CRWstereotactic frame is then affixed to the cranial vault afterinfiltration of the pin insertion sites with 1% lidocaine with 1/200,000epinephrine. Attention is directed to insure that the frame is nottilted or skewed for optimal imaging and target localization. Thepatient is then brought to the MRI scanner.

A mid-sagittal T1 weighted scout is obtained. This image is used toalign the gantry of the scanner so that axial images are parallel to theAC-PC (anterior commissure-posterior commissure) plane. Next T1 weighted(TR 400, TE 12/Fr, FOV 30×30, 2 NEX, 3 mm thickness) axial images areobtained through the basal ganglia. The patient is then taken to the CTscanner and after the appropriate localizer is placed, axial CT images(DFOV, 1.5 mm thickness) through the area of interest are obtained. Analternative imaging strategy involves first obtaining T1 weightedsagittal images followed by fast spin echo inversion recovery axial andcoronal images. This sequence is better delineates gray/white matterdifferences and allows for better visualization of the internal capsuleand optic tract. Another imaging the strategy is to use a 3 dimensionalSPGR volumetric sequence but these series generally require 10-11minutes and are thus subject to movement artifacts. These images permitreconstruction of the images in sagittal, coronal and axial planes with0.75-1.5 mm slice thickness.

The selected pallidal target based upon the location of the identifiedfocal epileptogenic nucleus lies 2-3 mm in front of the mid-commissuralpoint, 5-6 mm below the intercommisural line, and 19-22 mm lateral tothe midline of the third ventricle. The appropriate coordinates areobtained from both the MRI and CT images and are compared for accuracy.A correctly placed toxin infusion will lie just behind the posteriormargin of the mammilary bodies and just superior and lateral to theoptic tract on the appropriate images.

Once the imaging is complete, the patient is brought to the operatingroom. A single dose of an appropriate prophylactic antibiotic (typicallycefazolin) is given. The patient is placed in a supine semi-sittingposition, with the head slightly raised above the horizontal, and theframe is affixed to the table. The head of the table is not beexcessively elevated so as to prevent air embolism. Great care is takento insure that the patient is comfortable since his cooperation isnecessary for a successful procedure. A grounding patch is attached tothe patient to allow for stimulation and toxin lesioning. A small patchof hair is shaved over the appropriate frontal region and the area isthen prepared and draped. Draping is kept to a reasonable minimum toallow for intra-operative assessment of the patient. A single clearplastic drape is placed over the patient's head.

The skin is infiltrated with 1% lidocaine with 1/200,000 epinephrine. A2.5 cm parasagittal incision is made centered at a point about 3 cmlateral to the midline, in the midpupillary line, and 1-2 cm anterior tothe coronal suture. A Hudson brace is used to make a 1 cm burr hole andthe bone edges are waxed. The dura is coagulated and opened. Next, thepia of the underlying cortex is coagulated with the bipolar, and a smallpial incision is made to allow for atraumatic introduction of theelectrode. At this point the stereotactic frame is brought into positionand the electrode guide is lowered into the burr hole. The trajectory ofthe electrode subtends an angle of 65-70 degrees from the horizontal and5-10 degrees from the sagittal planes. A piece of Gelfoam is placedaround the guide tube and the skin is temporarily closed over the holewith nylon sutures to prevent excessive loss of CSF and brain settling.

Microelectrode recordings are made to better identify the optimal toxininfusion location and to minimize the risk of injury to the internalcapsule or the optic tract. The techniques for microelectrode recordingin the globus pallidus have been well described by Lozano et al (LozanoA, et al., Methods for microelectrode guided posteroventral pallidotomy,J Neurosurg 1996; 84:194-202). Tungsten microelectrodes with animpedance of 1-2 M Ohm at 1 khz are used.

The electrodes are clamped to the stereotactic frame and advancedthrough a guide tube with a microdrive. The microelectrode is connectedvia short leads to a preamplifier which increases the signal to noiseratio. The leads from the electrode to the preamplifier are kept shortto minimize the introduction of background electrical noise. The signalfrom the preamplifier is then filtered, amplified, and passed through awindow discriminator. The window discriminator is an electronic devicewhich converts action potentials to digital pulses. These digital pulsesare stored and analyzed off-line. In addition, the pulses can beconverted to an audio signal which is useful for listening to theactivity of cells without interference from background neuronal noise.

During pallidotomy, recordings proceed from the putamen and Gpe throughthe Gpi and then near the optic tract. Once the electrode emerges fromthe ventral border of the GP into the white matter of the ansalenticularis, neuronal activity diminishes along with backgroundactivity. Deeper penetration of 1-2 mm places the electrode tip in closeproximity to or within the optic tract. It is difficult to recorddirectly from the optic tract because it contains only axons and theaction potentials are correspondingly small. Photic stimulation withaveraging of the evoked visual potential is therefore used. Proximity tothe optic tract is determined by microstimulation as follows. A 1 sectrain consisting of 2 msec square wave pulses at 300 Hz is used toelicit visual phenomenon. Visual thresholds at or near the optic tractare usually between 2-20 æA. The patient report seeing flashing lightsof various colors or scotomata in the contralateral visual field.

Macrostimulation is also used to verify the location of the site fortoxin infusion. Impedance monitoring, stimulation, but not lesioning,are handled by a Radionics RF lesion generator. A macroelecrode with a2×1.6 mm uninsulated tip is introduced through the guide tube underimpedance monitoring. The impedance is seen to drop about 100 when thegray matter of the basal ganglia is reached. The electrode is stopped ata point 6 mm above the target and macrostimulation is then used tofurther delineate the optimal target location. Low frequency stimulationis performed with square wave pulses at a frequency of 2 Hz at 0-5 Voltsto obtain motor thresholds in order to insure that the lesion does notinjure the internal capsule. High frequency stimulation using squarewave pulses of 50-75 Hz at 0-5 Volts is used to assess for proximity tothe optic tract, speech dysfunction, and amelioration of symptoms.Stimulation is carried out at 6 mm, 4 mm, and 2 mm above the target andat the target. At each point both low and high frequency stimulation isperformed. To obtain the motor thresholds, low frequency stimulation isused and the voltage is gradually increased until fine contractions canbe seen in the contralateral hand and/or the tongue. The voltage atwhich definite contractions can be first seen is the motor threshold. Asthe electrode is lowered the thresholds are seen to decrease. The motorthresholds are around 4-5 volts at the highest electrode position anddecrease to about 2-3 volts at the target. When the electrode is nearthe target it is usually wise to perform high frequency stimulationfirst to insure that the electrode is not too close to the optic tractbefore proceeding with low frequency stimulation. Once the electrode is2 mm above target, visual thresholds are obtained by turning off theroom lights and asking the patient to report if he sees any flashinglights as the voltage as quickly increased and decreased with the highfrequency stimulation. The classical response is a perception offlashing lights or phosphenes in the contralateral hemifield. Theminimal voltage which elicits visual phenomenon constitutes the visualthreshold. The electrode is then lowered to the target position andvisual thresholds are again assessed. Correct placement of the electrodeis determined by visual thresholds between 2-3 volts.

Once the target location is verified A botulinum toxin type A can beinjected into the target tissue through a stereotactically placed 30gauge stainless steel tube using the method set forth by Levy R., etal., Lidocaine and muscimol microinjections in subthalamic nucleusreverse parkinsonian symptoms, Brain (2001), 124, 2105-2118. Thus, theinjection cannula can be connected to a 10-15 cm piece of polyethylenetubing with a inside diameter of 0.58 mm and sealed with epoxy glue. Theaqueous toxin can be preloaded in the cannula and polyethylene tubing.

After toxin infusion the patient is then assessed for any evidence ofmotor, sensory, visual, or speech impairment. If there are no deficits,the full complement of toxin is infused. With careful attention todetail the technique described above can be reliably used to create atoxin induced lesions in the posteroventral pallidum. Typically, theseacute lesions are about 100-150 cu mm and shrink over time.

At the completion of surgery the patient is allowed to take his nextscheduled medication. After a brief period of observation, the patientis returned directly to his room. An MRI scan is obtained within thefirst 24 hours to assess lesion location and to rule out unforeseencomplications. The patient continues with his preoperative medicationsand outside of mild analgesics, no other medications are necessary. Theintracranial botulinum toxin therapy cures the patient as he remainsfree of any epileptic seizures for between six months and five years.

Example 13 Treatment of Epilepsy by Administration of a Botulinum Toxinto an Epileptogenic Focus Located in the Left Hippocampus

The methodology set forth in Example 11 can be carried out to treatmesial temporal lobe epilepsy wherein the identified epileptogenic focusis located in the left hippocampus region of the brain by administeringthe botulinum toxin to the following stereotactic coordinates (in mm)(where the center of gravity is determined according to Talairach J., etal., Co-Planar Stereotactic Atlas of the Human Brain. 3-Dimensionalproportional system: an approach to cerebral imaging. Theime Verlag,Stuttgart, N.Y. (1988)): x −20, y −21, z −16. This intracranialbotulinum toxin therapy can cure the patient as he remains free of anyepileptic seizures for between six months and five years.

Example 14 Treatment of Epilepsy by Administration of a Botulinum Toxinto an Epileptogenic Focus Located in the Left Middle Temporal Gyrus

The methodology set forth in Example 11 can be carried out to treatmesial temporal lobe epilepsy wherein the identified epileptogenic focusis located in the left middle temporal gyrus region of the brain byadministering the botulinum toxin to the following stereotacticcoordinates (in mm) (where the center of gravity is determined accordingto Talairach J., et al., Co-Planar Stereotactic Atlas of the HumanBrain. 3-Dimensional proportional system: an approach to cerebralimaging. Theime Verlag, Stuttgart, N.Y. (1988)): x −52, y −19, z −10.This intracranial botulinum toxin therapy can cure the patient as heremains free of any epileptic seizures for between six months and fiveyears.

Example 15 Treatment of Epilepsy by Administration of a Botulinum Toxinto an Epileptogenic Focus Located in the Left Fusiform Gyrus

The methodology set forth in Example 11 can be carried out to treatmesial temporal lobe epilepsy wherein the identified epileptogenic focusis located in the left fusiform gyrus region of the brain byadministering the botulinum toxin to the following stereotacticcoordinates (in mm) (where the center of gravity is determined accordingto Talairach J., et al., Co-Planar Stereotactic Atlas of the HumanBrain. 3-Dimensional proportional system: an approach to cerebralimaging. Theime Verlag, Stuttgart, N.Y. (1988)): x −47, y −42, z −16.This intracranial botulinum toxin therapy can cure the patient as heremains free of any epileptic seizures for between six months and fiveyears.

Example 16 Treatment of Epilepsy by Administration of a Botulinum Toxinto an Epileptogenic Focus Located in the Right Hippocampus

The methodology set forth in Example 11 can be carried out to treatmesial temporal lobe epilepsy wherein the identified epileptogenic focusis located in the right hippocampus region of the brain by administeringthe botulinum toxin to the following stereotactic coordinates (in mm)(where the center of gravity is determined according to Talairach J., etal., Co-Planar Stereotactic Atlas of the Human Brain. 3-Dimensionalproportional system: an approach to cerebral imaging. Theime Verlag,Stuttgart, N.Y. (1988)): x +29, y −18, z −16. This intracranialbotulinum toxin therapy can cure the patient as he remains free of anyepileptic seizures for between six months and five years.

Example 17 Treatment of Epilepsy by Administration of a Botulinum Toxinto an Epileptogenic Focus Located in the Right Middle Temporal Gyrus

The methodology set forth in Example 11 can be carried out to treatmesial temporal lobe epilepsy wherein the identified epileptogenic focusis located in the right middle temporal gyrus region of the brain byadministering the botulinum toxin to the following stereotacticcoordinates (in mm) (where the center of gravity is determined accordingto Talairach J., et al., Co-Planar Stereotactic Atlas of the HumanBrain. 3-Dimensional proportional system: an approach to cerebralimaging. Theime Verlag, Stuttgart, N.Y. (1988)): x +59, y −22, z −4.This intracranial botulinum toxin therapy can cure the patient as heremains free of any epileptic seizures for between six months and fiveyears.

Example 18 Treatment of Epilepsy by Administration of a Botulinum Toxinto an Epileptogenic Focus Located in the Right Superior Temporal Gyrus

The methodology set forth in Example 11 can be carried out to treatmesial temporal lobe epilepsy wherein the identified epileptogenic focusis located in the right superior temporal gyrus region of the brain byadministering the botulinum toxin to the following stereotacticcoordinates (in mm) (where the center of gravity is determined accordingto Talairach J., et al., Co-Planar Stereotactic Atlas of the HumanBrain. 3-Dimensional proportional system: an approach to cerebralimaging. Theime Verlag, Stuttgart, N.Y. (1988)): x +64, y −27, z +11.This intracranial botulinum toxin therapy can cure the patient as heremains free of any epileptic seizures for between six months and fiveyears.

Example 19 Treatment of Epilepsy by Administration of a Botulinum Toxinto an Epileptogenic Focus Located in the Right Fusiform Gyrus

The methodology set forth in Example 11 can be carried out to treatmesial temporal lobe epilepsy wherein the identified epileptogenic focusis located in the right fusiform gyrus region of the brain byadministering the botulinum toxin to the following stereotacticcoordinates (in mm) (where the center of gravity is determined accordingto Talairach J., et al., Co-Planar Stereotactic Atlas of the HumanBrain. 3-Dimensional proportional system: an approach to cerebralimaging. Theime Verlag, Stuttgart, N.Y. (1988)): x +44, y −57, z −16.This intracranial botulinum toxin therapy can cure the patient as heremains free of any epileptic seizures for between six months and fiveyears.

Example 20 Treatment of Epilepsy by Administration of a Botulinum Toxinto an Epileptogenic Focus Located in the Right Mesial Frontal Gyrus

The methodology set forth in Example 11 can be carried out to treatmesial temporal lobe epilepsy wherein the identified epileptogenic focusis located in the right mesial frontal gyrus region of the brain byadministering the botulinum toxin to the following stereotacticcoordinates (in mm) (where the center of gravity is determined accordingto Talairach J., et al., Co-Planar Stereotactic Atlas of the HumanBrain. 3-Dimensional proportional system: an approach to cerebralimaging. Theime Verlag, Stuttgart, N.Y. (1988)): x +10, y +41, z +14.This intracranial botulinum toxin therapy can cure the patient as heremains free of any epileptic seizures for between six months and fiveyears.

The Examples above show that method within the scope of the claims canbe used to successfully treat epilepsy in humans regardless of theparticular type or origin of the epilepsy. The invention thereforeprovides a treatment for many different types of epilepsy.

Methods according to the present invention can also be used diversemovement disorders, including essential tremor, multiple sclerosisrelated tremors, post traumatic tremors, post hemiplegic tremors,parkinsonian tremors and epilepsy.

An intracranial neurotoxin administration method for treating a movementdisorder according to the invention disclosed herein for has manybenefits and advantages, including the following:

1. the symptoms of a movement disorder can be dramatically reduced.

2. the symptoms of a movement disorder can be reduced for from about twoto about four months per injection of neurotoxin and for from about oneyear to about five years upon use of a controlled release neurotoxinimplant.

3. the injected or implanted neurotoxin exerts an intracranial targettissue site specific tremor suppressant effect.

4. the injected or implanted neurotoxin shows little or no tendency todiffuse or to be transported away from the intracranial injection orimplantation site.

5. few or no significant undesirable side effects occur fromintracranial injection or implantation of the neurotoxin.

6. the amount of neurotoxin injected intracranially can be considerablyless than the amount of the same neurotoxin required by other routes ofadministration (i.e. intramuscular, intrasphincter, oral or parenteral)to achieve a comparable tremor suppressant effect.

7. the tremor suppressant effects of the present methods can result inthe desirable side effects of greater patient mobility, a more positiveattitude, and an improved quality of life.

8. high, therapeutic doses of a neurotoxin can be delivered to anintracranial target tissue over a prolonged period without systemictoxicity.

Although the present invention has been described in detail with regardto certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of neurotoxins can be effectively used inthe methods of the present invention. Additionally, the presentinvention includes intracranial administration methods wherein two ormore neurotoxins, such as two or more botulinum toxins, are administeredconcurrently or consecutively. For example, botulinum toxin type A canbe administered intracranially until a loss of clinical response orneutralizing antibodies develop, followed by administration of botulinumtoxin type B. Alternately, a combination of any two or more of thebotulinum serotypes A-G can be intracranially administered to controlthe onset and duration of the desired therapeutic result. Furthermore,non-neurotoxin compounds can be intracranially administered prior to,concurrently with or subsequent to administration of the neurotoxin toproved adjunct effect such as enhanced or a more rapid onset of tremorsuppression before the neurotoxin, such as a botulinum toxin, begins toexert its more long lasting tremor suppressant effect.

Our invention also includes within its scope the use of a neurotoxin,such as a botulinum toxin, in the preparation of a medicament for thetreatment of a movement disorder, by intracranial administration of theneurotoxin.

All references, articles, patents, applications and publications setforth above are incorporated herein by reference in their entireties.

Accordingly, the spirit and scope of the following claims should not belimited to the descriptions of the preferred embodiments set forthabove.

1. A method for treating focal epilepsy in a patient in need thereof,the method comprising the steps of: identifying an epileptogenic focusof a patient; and intracranially administering a botulinum toxin to theepileptogenic focus of the patient, thereby treating the focal epilepsyof the patient.
 2. The method of claim 1, wherein the botulinum toxin isselected from the group consisting of botulinum toxin types A, B, C₁, D,E, F and G.
 3. The method of claim 1, wherein the botulinum toxin isbotulinum toxin type A.
 4. The method of claim 1, wherein the botulinumtoxin is administered in an amount of between about 10⁻³ U/kg and about10 U/kg of patient weight.
 5. The method of claim 1, wherein the methodalleviates epilepsy for between about 1 month and about 5 years.
 6. Themethod of claim 1, wherein the identifying step utilizeselectroencephalography to determine the location of the epileptogenicfocus.
 7. The method of claim 1, wherein the botulinum toxin isadministered to an epileptogenic focus at a temporal gyrus.
 8. Themethod of claim 1, wherein the botulinum toxin is administered to anepileptogenic focus at a fusiform gyrus.
 9. The method of claim 1,wherein the botulinum toxin is administered to an epileptogenic focus ata middle or superior temporal gyrus.
 10. The method of claim 1 whereinthe botulinum toxin is administered to an epileptogenic focus a left orright or fusiform gyrus.
 11. The method of claim 1 wherein the botulinumtoxin is administered to an epileptogenic focus at a mesial region of atemporal lobe.
 12. The method of claim 1, wherein the intracranialadministration step comprises implantation of a controlled releasebotulinum toxin system.
 13. A method for treating focal epilepsy in apatient in need thereof, the method comprising the step of intracranialadministration of a therapeutically effective amount of a botulinumtoxin type A to an epileptogenic focus of a patient, thereby treatingfocal epilepsy of the patient.
 14. The method of claim 13, wherein thepatient experiences intractable seizures as a result of the focalepilepsy.
 15. The method of claim 13, wherein the botulinum toxin isadministered in an amount of between about 10⁻² U/kg and about 1 U/kg ofpatient weight.
 16. A method for treating focal epilepsy, the comprisingthe step of administering intracranially a therapeutically effectiveamount of a botulinum toxin to an identified epileptogenic focus,wherein the identified epileptogenic focus is located in an anteriorportion of the temporal lobe, thereby treating the focal epilepsy. 17.The method of claim 16, wherein the anterior portion is 5 to 6 cm fromthe tip of the temporal lobe along a middle temporal gyrus.
 18. Themethod of claim 16, wherein the botulinum toxin is selected from thegroup consisting of botulinum toxin types A, B, C₁, D, E, F and G. 19.The method of claim 16, wherein the botulinum toxin is botulinum toxintype A.
 20. The method of claim 16, wherein the botulinum toxin isadministered in an amount of between about 10⁻³ U/kg and about 100 U/kgof patient weight.